KR20240132405A - Composition for improvement, treatment or prevention of muscular disorders, or improvement of muscular functions comprising melinjo seed - Google Patents

Abstract

The present invention relates to a composition containing melinjo seeds, such as melinjo seed pulverized product or extract, as an active ingredient, for preventing and treating muscle diseases or improving muscle function. Melinjo seeds, such as melinjo seed pulverized product or melinjo seed extract according to the present invention, reduce the mRNA expression of MuRF-1 and atrogin-1 involved in muscle protein degradation, increase the activity of mTOR involved in muscle protein synthesis, and increase muscle function and muscle mass, thereby being useful for preventing, improving, or treating various muscle diseases, such as sarcopenia, muscular atrophy, muscular dystrophy, atony, muscle degeneration, myasthenia, and cachexia.

Description

Composition for improvement, treatment or prevention of muscular disorders, or improvement of muscular functions comprising melinjo seed as an effective ingredient

The present invention relates to a composition for improving, treating or preventing muscle diseases, or for improving muscle function, and more particularly, to a composition for improving, treating or preventing muscle diseases, or for improving muscle function, containing melinjo seeds, such as melinjo seed powder or melinjo seed extract, as an active ingredient.

Muscle atrophy is caused by a gradual decrease in muscle mass, and refers to muscle weakness and degeneration (Cell, 119(7): 907-910, 2004). Muscle atrophy is promoted by inactivity, oxidative stress, and chronic inflammation, and impairs muscle function and exercise capacity (Clinical Nutrition, 26(5): 524-534, 2007). The most important factor determining muscle function is muscle mass, which is maintained by the balance between protein synthesis and degradation. Muscle atrophy occurs when protein degradation exceeds synthesis (The International Journal of Biochemistry and Cell Biology, 37(10): 1985-1996, 2005).

Muscle size is regulated by intracellular signaling pathways that induce anabolism or catabolism within the muscle. When signaling reactions that induce synthesis rather than breakdown of muscle proteins occur more frequently, muscle protein synthesis increases, which is manifested as an increase in muscle size (hypertrophy) or an increase in the number of muscle fibers (hyperplasia) due to an increase in muscle protein (The Korea Journal of Sports Science, 20(3): 1551-1561, 2011).

Factors involved in muscle protein synthesis induce protein synthesis by phosphorylating downstream proteins starting from stimulation of the phosphatidylinositol-3 kinase (PI3K)/Akt pathway in muscle cells. Mammalian target of rapamycin (mTOR) activation by PI3K/Akt signaling is recognized as a central growth signaling factor that integrates various growth signals in cells. mTOR induces muscle protein synthesis by activating two factors that initiate mRNA translation, 4E-binding protein (4EBP1) and phosphorylated 70-kDa ribosomal S6 kinase (p70S6K), thereby contributing to the increase in muscle mass (The Korea Journal of Sports Science, 20(3): 1551-1561, 2011; The International Journal of Biochemistry and Cell Biology, 43(9): 1267-1276, 2011). On the other hand, when the transcription factor forhead box (FoxO) moves from the cytoplasm to the nucleus, it increases the expression of E3 ubiquitin ligase factors atrogin-1 and MuRF1 involved in protein degradation (Disease Models and Mechanisms, 6: 25-39, 2013). When their expression levels increase, protein degradation in muscles is promoted, resulting in a decrease in muscle mass. Therefore, the promotion of mTOR activity and the suppression of atrogin-1 and MuRF1 expression increase the amount of muscle protein, thereby increasing muscle mass.

Differentiation of muscle cells and muscle formation are regulated by various muscle regulatory factors. Among them, induction of myogenin expression by MyoD activity is the most important factor in the fusion of myoblasts and is involved in the formation of myotubes. Muscle fibers formed through this process form bundles and ultimately form muscles (Cellular and Molecular Life Sciences, 70: 4117-4130, 2013).

Melinjo ( Gnetum gnemon Linn.) is a plant belonging to the Gnetaceae family. Its scientific name is Gnetum gnemon Linn. Melinjo is mainly produced in Thailand, Malaysia, Indonesia, and Fiji, and its leaves and seeds are mainly used for food. Various physiological activities such as antioxidant, anti-inflammatory, antibacterial, anticancer, antihypertensive, and antidiabetic activities have been reported for Melinjo seeds (Journal of Plant Biochemistry and Biotechnology 20(2): 234-24, 2011; International Journal of Pharmacognosy and Phytochemical Research 7(3): 531-539, 2015; Food Sci. Technol, Campinas, 42, e100121, 2022).

However, prior to the present invention, it was not known whether Melinzo seeds could prevent and treat muscle diseases or improve muscle function.

Accordingly, the inventors of the present invention searched for a natural substance that has excellent muscle function regulating activity and can be safely applied, and as a result, confirmed that melinzo seed, such as melinzo seed powder or melinzo seed extract, has activity for improving, treating or preventing muscle diseases, or improving muscle function, thereby completing the present invention.

Therefore, the purpose of the present invention is to provide a food composition for improving or preventing muscle disease or improving muscle function, containing melinzo seeds as an effective ingredient, such as melinzo seed powder or melinzo seed extract.

Another object of the present invention is to provide a pharmaceutical composition for treating or preventing muscle disease containing melinzo seeds, such as melinzo seed powder or melinzo seed extract, as an active ingredient.

Another object of the present invention is to provide a cosmetic composition for improving or preventing muscle disease or improving muscle function, containing melinzo seed as an active ingredient, such as melinzo seed powder or melinzo seed extract.

Another object of the present invention is to provide a feed additive for improving or preventing muscle disease or improving muscle function, which contains melinzo seed as an effective ingredient, such as melinzo seed powder or melinzo seed extract.

To achieve the above purpose, the present invention provides a food composition for improving or preventing muscle disease or improving muscle function, containing melinzo seed as an effective ingredient, such as melinzo seed powder or melinzo seed extract.

In addition, the present invention provides a pharmaceutical composition for treating or preventing muscle disease containing melinzo seeds as an active ingredient, such as melinzo seed powder or melinzo seed extract.

In addition, the present invention provides a cosmetic composition for improving or preventing muscle disease or improving muscle function, containing melinzo seed as an active ingredient, such as melinzo seed powder or melinzo seed extract.

In addition, the present invention provides a feed additive for improving or preventing muscle disease or improving muscle function, containing melinzo seed as an effective ingredient, such as melinzo seed powder or melinzo seed extract.

In addition, the present invention provides a method for treating muscle disease by administering the composition to a human or an animal other than a human.

Furthermore, the present invention provides novel uses of melinzo seeds, such as melinzo seed powder or melinzo seed extract, for the manufacture of a medicament for treating muscle diseases.

Melinzo seeds, such as the melinzo seed powder or melinzo seed extract according to the present invention, have an excellent effect in inhibiting the mRNA expression of MuRF1 and atrogin-1 involved in muscle protein decomposition and increasing the activity of mTOR involved in muscle protein synthesis. In addition, by increasing muscle function and muscle mass, it can prevent, treat or improve muscle function decline, muscle loss, etc. caused by various diseases. Since the present invention is a natural product, it can be used safely without side effects and can be usefully used in the manufacture of foods, pharmaceuticals, cosmetics or feed additives.

Figure 1 shows the results of confirming the effect of treatment with the hot water extract of Melinzo seeds of Example 1 on reducing the mRNA expression levels of MuRF1 and atrogin-1 in C2C12 muscle cells.
Figure 2 shows the results of confirming the effect of increasing mTOR activity according to treatment with the melinzo seed water extract of Example 1 in C2C12 muscle cells. Each graph represents the mean value (mean±SEM, n=3). ** indicates a significant difference at p<0.01 compared to the control group (Duncan's test).

Hereinafter, the composition of the present invention will be described in detail.

The present invention provides a food composition containing melinzo seeds, such as melinzo seed powder or melinzo seed extract, for improving or preventing muscle disease, or for improving muscle function; a pharmaceutical composition containing melinzo seeds, such as melinzo seed powder or melinzo seed extract, for treating or preventing muscle disease; or a cosmetic composition containing melinzo seeds, such as melinzo seed powder or melinzo seed extract, for improving or preventing muscle disease, or for improving muscle function; or a feed additive containing melinzo seeds, such as melinzo seed powder or melinzo seed extract, for improving or preventing muscle disease, or for improving muscle function; or a method for treating muscle disease comprising applying melinzo seeds, such as melinzo seed powder or melinzo seed extract, to a human or a non-human mammal.

In this specification, the 'Melinzo seeds' may use the melinzo seed powder or the melinzo seed extract. In the present invention, the melinzo seeds may be purchased and used commercially, or may be collected or grown in nature.

In this specification, the 'melinzo seed pulverized product' can be used by preparing dried melinzo seeds in a form that can be easily ingested by mammals, including humans, according to a method that can be easily implemented by a person of ordinary skill in the art to which the present invention pertains, and in which the active ingredient can be easily released from the dried pulverized product in the intestines after ingestion, thereby being easily absorbed into the mammalian body. The shape or size of the dried pulverized product particles for this purpose is not limited, but it is preferable to prepare it in the form of a fine powder as much as possible because the release of the active ingredient described above is facilitated as the surface area of the particles is maximized. In one embodiment of the present invention, the pulverized product was prepared by pulverizing dried melinzo seeds in a mixer.

In this specification, the 'Melinzo seed extract' is obtained by extracting dried Melinzo seeds using a suitable solvent, and includes all forms of an extract, a diluted or concentrated extract, a dried product obtained by drying the extract, or a controlled or purified product thereof.

Specifically, the melinzo seed extract may be obtained by extracting melinzo seeds with at least one solvent selected from water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid and a supercritical fluid. The organic solvent having 1 to 6 carbon atoms is preferably at least one selected from alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane and petroleum ether, but is not limited thereto. More preferable solvents for extracting the melinzo seed extract of the present invention are water, ethanol, ethyl acetate, hexane, a subcritical fluid or a supercritical fluid.

As a specific example of the present invention, the melinzo seeds may be prepared by mixing with an extraction solvent at a weight ratio of 1:2 to 20, preferably 1:5 to 15, and more preferably 1:8 to 12 to prepare an extract. If the weight ratio of the melinzo seeds and the extraction solvent is out of the above range, the effective ingredient of the melinzo seeds may be extracted in a small amount in the extract.

As another specific example, the melinzo seed extract of the present invention can be obtained by extracting melinzo seeds under ultra-high pressure conditions of 100 MPa or more. If necessary, it can be prepared by additionally including filtration and concentration steps according to methods known in the art.

The above extraction can be performed by a heating extraction method, a cold immersion extraction method, a reflux cooling extraction method, an ultrasonic extraction method, an ultrasonic pressure extraction method, a subcritical fluid extraction method, a supercritical fluid extraction method, etc., and there is no limitation as long as it is an extraction method obvious to those skilled in the art. The extraction can be performed at room temperature, but for more efficient extraction, it can be performed under heated conditions, and the extraction can be performed at a temperature of 20 to 100°C, preferably about 30 to 100°C, but is not limited thereto. The extraction time can be performed for 1 to 10 hours, preferably 2 to 8 hours, more preferably 3 to 5 hours, but is not limited thereto, and may vary depending on conditions such as the extraction solvent and the extraction temperature. The extraction can be performed once or more multiple times in order to obtain a larger amount of the active ingredient, and can be repeatedly extracted preferably 1 to 5 times, more preferably 2 to 3 times.

The above melinzo seed extract may be a crude extract of melinzo seeds, or may be a solvent-soluble fraction obtained by re-fractionating the crude extract with a second solvent. The extract extracted by the above method or the soluble fraction of the extract may be used as is, but may be used in the form of an extract after filtration and concentration, and may be manufactured into a powder form and used by an additional process such as distillation under reduced pressure, freeze drying, or spray drying after concentration.

In this specification, the term 'fraction' refers to a result obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various components. In the present invention, it refers to a result obtained by a fractionation method for separating a specific component or a specific group from a melinzo seed extract prepared as described above.

In order to obtain the melinzo seed fraction according to the present invention, conventional fractionation solvents known in the art, for example, polar solvents such as water, ethanol, methanol, anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms, and nonpolar solvents such as hexane, butanol, ethyl acetate, chloroform, dichloromethane, or mixed solvents thereof, can be used, but are not limited thereto.

The melinzo seed fraction of the present invention may also include fractions obtained by additionally applying a purification process. For example, fractions obtained by passing the melinzo seed extract according to the present invention through an ultrafiltration membrane having a predetermined molecular weight cut-off value, fractions obtained by additionally performing various purification methods such as separation by various chromatographies (designed for separation by size, charge, hydrophobicity or affinity), etc. are also included in the melinzo seed extract of the present invention.

In addition, a fermentation product or an extract of a fermentation product obtained by adding microorganisms (bacteria, yeast mold, etc.) applicable to the human body to the melinzo seed powder or melinzo seed extract can also be included in the melinzo seed extract.

In this specification, 'muscle' comprehensively refers to tendons, muscles, and tendons, and 'muscle function' means the ability of a muscle to exert force by contraction, and includes muscle strength, which is the ability of a muscle to exert maximum contraction force to overcome resistance, muscle endurance, which is the ability of a muscle to repeat contraction and relaxation for a given weight for a long time or for a number of times, and explosiveness, which is the ability to exert strong force in a short period of time. The term 'improvement of muscle function' as used in this specification means further improving muscle function by increasing muscle mass.

In a specific embodiment of the present invention, the inventors prepared a melinzo seed extract by extracting melinzo seeds using hot water, ethanol, ethyl acetate or hexane as a solvent, or by using ultra-high pressure extraction, subcritical fluid extraction or supercritical fluid extraction (Examples 1 to 7).

As a result of treating C2C12 muscle cells with the melinzo seed extract manufactured as above, it was confirmed that the mRNA expression of MuRF-1 and atrogin-1 involved in muscle protein degradation was significantly inhibited (Fig. 1, Table 1). In addition, as a result of treating C2C12 muscle cells with the melinzo seed extract, it was confirmed that the activity of mTOR involved in muscle protein synthesis was significantly increased (Fig. 2, Table 2). When treating the melinzo seed powder or melinzo seed extract using an immobilization muscle atrophy animal model using a skin stapler, muscle strength and muscle weight were significantly increased compared to the muscle atrophy group, respectively (Table 3).

If the composition of the present invention is a food composition for improving and preventing muscle disease, or improving muscle function, it can be used for preventing or improving muscle disease caused by muscle wasting or degeneration. Muscle wasting and degeneration are caused by genetic factors, acquired factors, aging, etc., and muscle wasting is characterized by gradual loss of muscle mass, weakening and degeneration of muscles, especially skeletal muscles or voluntary muscles and cardiac muscles. Examples of diseases related thereto include sarcopenia, muscular atrophy, muscular dystrophy, atony, muscle degeneration, myasthenia, cachexia, etc. The composition of the present invention has a muscle mass increasing effect, and the type of muscle is not limited.

In this specification, 'prevention' means anything that suppresses or delays the above muscle disease.

The term 'improvement' of the present invention means at least reducing the degree of a parameter, for example, a symptom, associated with a condition being treated by administration of a composition comprising the ground melinzo seed or melinzo seed extract of the present invention.

The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food, food additive, and feed, and is intended for consumption by humans or animals including livestock. The food composition of the above type can be manufactured in various forms according to conventional methods known in the art.

For example, as a health food, the melinzo seed powder or melinzo seed extract of the present invention can be manufactured in the form of tea, juice, and drinks for consumption, or can be granulated, encapsulated, and powdered for consumption.

In addition, functional foods can be produced by adding the pulverized melinzo seed or melinzo seed extract of the present invention to beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled fruits, jams, marmalade, etc.), fish, meat and processed foods thereof (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g., butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauce, etc.).

In addition, the melinzo seed powder or melinzo seed extract of the present invention can be manufactured and used in the form of a powder or concentrate for use as a food additive.

Melinzo seed powder or melinzo seed extract can be contained as an effective ingredient of a food composition for improving and preventing muscle disease or improving muscle function, and the amount thereof is not particularly limited to an effective amount for improving and preventing muscle disease or improving muscle function, but is preferably 0.01 to 100 wt% based on the total weight of the entire composition. The food composition of the present invention can be prepared by mixing melinzo seed powder or melinzo seed extract with another active ingredient known to be effective in improving and preventing muscle disease or improving muscle function.

In addition, when the composition of the present invention is a pharmaceutical composition for treating or preventing muscle disease, it can be used for preventing or treating muscle disease caused by muscle wasting or degeneration. Muscle wasting and degeneration are caused by genetic factors, acquired factors, aging, etc., and muscle wasting is characterized by gradual loss of muscle mass, weakening and degeneration of muscles, especially skeletal muscles or voluntary muscles and cardiac muscles. Examples of diseases related thereto include sarcopenia, muscular atrophy, muscular dystrophy, hypotonia, muscle degeneration, myasthenia gravis, cachexia, etc. The composition of the present invention has an effect of increasing muscle mass, and the type of muscle is not limited.

In this specification, 'prevention' means anything that inhibits or delays the above muscle disease.

In this specification, 'treatment' means reversing, alleviating, inhibiting the progression of, or preventing the symptoms of the muscle disease.

The melinzo seed powder or melinzo seed extract according to the present invention can be used as is or in a pharmaceutically acceptable form for the purpose of treating or preventing muscle diseases. The term "pharmaceutically acceptable" as used herein refers to a non-toxic composition that is physiologically acceptable and does not inhibit the action of an active ingredient when administered to a human and does not typically cause allergic reactions such as gastrointestinal disorders or dizziness or similar reactions.

The pharmaceutical composition of the present invention may contain a pharmaceutically effective amount of a powdered melinzo seed or a melinzo seed extract alone or may contain one or more pharmaceutically acceptable carriers. The term "pharmaceutically effective amount" as used herein refers to an amount that exhibits a greater response than that of a negative control group, and preferably refers to an amount sufficient to treat or prevent muscle disease.

The pharmaceutical composition of the present invention may contain 0.01 to 99.99 wt% of crushed melinzo seed or melinzo seed extract, and the remainder may be comprised of a pharmaceutically acceptable carrier.

The carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes. Pharmaceutically acceptable carriers may be referred to those described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

Meanwhile, the route of administration of the pharmaceutical composition according to the present invention is not limited thereto, but may be administered orally or parenterally. Parenteral routes of administration include, for example, various routes such as transdermal, nasal, abdominal, intramuscular, subcutaneous, or intravenous.

In addition, the pharmaceutical composition of the present invention can be administered in combination with a known compound known to be effective in treating or preventing muscle diseases.

In addition, the composition for improving and preventing muscle disease or improving muscle function of the present invention may be a cosmetic composition. The cosmetic composition of the present invention contains a powdered melinzo seed or an extract of melinzo seed as an active ingredient, and may be manufactured in the form of a basic cosmetic composition (a cleanser such as a toner, a cream, an essence, a cleansing foam, and a cleansing water, a pack, a body oil), a color cosmetic composition (foundation, lipstick, mascara, makeup base), a hair product composition (shampoo, rinse, hair conditioner, hair gel), a soap, etc., together with a dermatologically acceptable excipient.

The above excipients may include, but are not limited to, skin softeners, skin penetration enhancers, colorants, fragrances, emulsifiers, thickeners, and solvents. In addition, fragrances, pigments, sterilizers, antioxidants, preservatives, moisturizers, etc. may be additionally included, and thickeners, inorganic salts, synthetic polymers, etc. may be included for the purpose of improving physical properties.

The content of the pulverized melinzo seed and the melinzo seed extract contained in the cosmetic composition of the present invention is not limited thereto, but is preferably 0.001 to 10 wt%, and more preferably 0.01 to 5 wt%, based on the total weight of the entire composition. If the content is less than 0.001 wt%, the desired effect cannot be expected, and if it exceeds 10 wt%, there may be difficulties in safety or formulation. The cosmetic composition of the present invention can be prepared by mixing the pulverized melinzo seed or the melinzo seed extract with other active ingredients known to be effective in improving and preventing muscle diseases, or improving muscle function.

In addition, when the composition of the present invention is a feed additive for improving and preventing muscle disease or improving muscle function, it can be used for preventing or improving muscle disease caused by muscle wasting or degeneration in animals.

In this specification, 'feed additive' includes a substance added to feed for various purposes such as nutrient supplementation and prevention of weight loss, promotion of digestibility and utilization of fiber in feed, improvement of milk quality, prevention of reproductive disorders and improvement of conception rate, and prevention of high temperature stress in summer. The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act, and may additionally include mineral preparations such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals; mineral preparations which are trace minerals such as zinc, copper, cobalt, and selenium; vitamins such as carotene, vitamins A, D, E, nicotinic acid, and vitamin B complex; protected amino acids such as methionine and lysine; protected fatty acids such as fatty acid calcium salts; probiotics (lactic acid bacteria), yeast cultures, and mold fermentations; and yeast agents.

In this specification, 'feed' means any natural or artificial diet, meal, etc., or a component of said meal, which is intended for eating, ingesting, and digesting by an animal, or suitable therefor. The feed containing the composition for treating or preventing muscle disease according to the present invention as an effective ingredient can be manufactured into various forms of feed known in the art, and preferably includes, but is not limited to, concentrated feed, forage, and/or special feed.

The feed additive for improving and preventing muscle disease or improving muscle function according to the present invention can be manufactured by adding crushed Melinzo seeds or Melinzo seed extract in an appropriate effective concentration range according to various feed manufacturing methods known in the art.

In addition, the present invention provides a method for treating muscle disease, comprising the step of administering to a mammal an effective amount of a pharmaceutical composition containing a powdered melinzo seed or a melinzo seed extract as an active ingredient.

The term 'mammal' as used herein refers to a mammal that is the subject of treatment, observation or experiment, preferably a human.

The term 'effective amount' as used herein means an amount of an effective ingredient or pharmaceutical composition that is thought by a researcher, veterinarian, physician or other clinician to induce a biological or medical response in a tissue, animal or human, including an amount that induces alleviation of symptoms of a disease or disorder. The effective amount and frequency of administration of the effective ingredient of the present invention may vary depending on the desired effect.

The pharmaceutically effective amount of the melinzo seed powder or melinzo seed extract according to the present invention is 0.001 to 100 mg/day/kg of body weight, preferably 0.01 to 10 mg/day/kg of body weight. However, the pharmaceutically effective amount may be adjusted according to various factors such as the disease and its severity, the patient's age, weight, health condition, sex, administration route, and treatment period.

Hereinafter, the melinzo seed powder or melinzo seed extract according to the present invention will be described in detail with examples and test examples.

<Example>

Example 1: Preparation of Melinzo seed hot water extract

Dried melinzo seeds were ground in a mixer, and 10 g of the ground melinzo seed sample was added to 100 mL of water and extracted at 80°C for 3 hours. The extracted sample was filtered under reduced pressure with a Whatman No. 1 filter, and the filtered extract was concentrated in a vacuum rotary evaporator to remove the solvent, thereby obtaining a melinzo seed hot water extract.

Example 2: Preparation of ethanol extract of Melinzo seeds

Dried melinzo seeds were ground in a mixer, and 10 g of the ground melinzo seed sample was added to 100 mL of 100% ethanol and extracted at 40°C for 3 hours. The extracted sample was filtered under reduced pressure through a Whatman No. 1 filter, and the filtered extract was concentrated in a vacuum rotary evaporator and the solvent was removed to obtain an ethanol extract of melinzo seeds.

Example 3: Preparation of ethyl acetate extract of Melinzo seeds

Dried melinzo seeds were ground in a mixer, and 10 g of the ground melinzo seed sample was added to 100 mL of ethyl acetate and extracted at 40°C for 3 hours. The extracted sample was filtered under reduced pressure through a Whatman No. 1 filter, and the filtered extract was concentrated in a vacuum rotary evaporator to remove the solvent, thereby obtaining melinzo seed ethyl acetate extract.

Example 4: Preparation of hexane extract of Melinzo seeds

Dried melinzo seeds were ground in a mixer, and 10 g of the ground melinzo seed sample was added to 100 mL of hexane and extracted at 40°C for 3 hours. The extracted sample was filtered under reduced pressure through a Whatman No. 1 filter, and the filtered extract was concentrated in a vacuum rotary evaporator to remove the solvent, thereby obtaining a melinzo seed hexane extract.

Example 5: Preparation of ultra-high pressure extract of Melinzo seeds

The dried melinzo seeds were ground in a mixer, and 1 g of the ground melinzo seeds and 76 mL of 18% (v/v) ethanol were placed in a polyethylene pack, sealed, and extracted using an ultra-high pressure extraction device (Frescal MFP-7000; Mitsubishi Heavy Industries). The ultra-high pressure extraction conditions were an extraction pressure of 320 MPa and an extraction time of 5 min. The extracted sample was filtered through a Whatman No. 2 filter, and the filtered extract was concentrated in a vacuum rotary evaporator to remove the solvent, thereby obtaining a melinzo seed ultra-high pressure extract.

Example 6: Preparation of subcritical extract of Melinzo seeds

The dried melinzo seeds were ground in a mixer, and 50 g of the ground melinzo seeds were placed in a subcritical water reactor of a subcritical extraction device (Biovan, Gyeonggi, Korea) with 1 L of water and sealed. After sealing, the temperature of the reactor was raised to 200°C, and when the temperature of the reactor reached 200°C, heating was stopped and the temperature was maintained for 20 minutes to perform extraction. After 20 minutes, the extract was transferred to a storage tank supplied with cooling water, rapidly cooled to 30°C, and centrifuged at 3,600 rpm for 30 minutes to separate the floating residue, and only the supernatant was collected. The melinzo seed subcritical extract was obtained by removing all the solvent using a freeze dryer (Ilshin Lab Co. Ltd., Seoul, Korea).

Example 7: Preparation of supercritical extract of Melinzo seeds

The dried melinzo seeds were ground in a mixer, and 10 g of the ground melinzo seeds were filled into a sample cartridge and extracted using a supercritical fluid extraction device (SFX 3560, Isco Inc., Lincoln, NE, USA). The supercritical extraction conditions were as follows: extraction pressure 300 bar, extraction temperature 50°C, supercritical carbon dioxide flow rate 60 mL/min, and extraction time 2 h. Upon completion of the supercritical fluid extraction, the pressure of the extraction device was lowered to release the supercritical fluid state, thereby obtaining the melinzo seed supercritical extract.

<Example of an exam>

Test Example 1: Inhibitory effect on expression of muscle protein degradation biomarker

C2C12 myoblasts (ATCC; Manassas, VA, USA) were seeded in 6-well plates at a density of 5 × 10 ⁵ cells/mL with Dulbecco's modified Eagle's media (DMEM; Hyclone) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). When the cell density reached approximately 80–85%, the medium in the well was removed, and the cells were treated with DMEM (Hyclone) containing 2% horse serum (HS; Hyclone) to induce myotube differentiation. The medium was replaced with fresh medium every two days, and differentiation was performed for a total of 4 days. After differentiation, 40 μg/mL and 80 μg/mL of the water extract of Example 1 and the ethanol extract of Example 2 were dissolved in DMEM (Hyclone) containing 10 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), respectively, and treated to the cells. After 24 hours, total RNA was isolated using TRIzol reagent (Takara, Otsu, Japan). The isolated total RNA was quantified using NanoDrop (NanoDrop 1000; Thermo Fisher Scientific Inc., Waltham, MA, USA). 16 μL of the quantified RNA was used to synthesize cDNA using Reverse Transcriptase Premix (ELPIS-Biotech) and a PCR machine (Gene Amp PCR System 2700; Applied Biosystems, Foster City, CA, USA) at 42°C for 55 minutes and 70°C for 15 minutes. Next, 1 μL of the generated 16 μL cDNA was amplified by PCR with PCR premix (ELPIS-Biotech) and the specific primers (Bioneer, Daejeon, Korea). Amplification was performed under the following conditions: pretreatment at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 58°C for 15 seconds, and extension at 72°C for 45 seconds, for a total of 35 cycles, followed by a final extension at 72°C for 5 minutes.

MuRF1

Forward primer: 5'-CCGGACGGAAATGCTATGGA-3' (SEQ ID NO: 1)

Reverse primer: 5'-AGCCTGGAAGATGTCGTTGG-3' (SEQ ID NO: 2)

Atrogin-1

Forward primer: 5'-GTTACTGCAACAAGGAGAATCTGTT-3' (SEQ ID NO: 3)

Reverse primer: 5'-CCGTATGAGTCTTATGTTTTGCTGG-3' (SEQ ID NO: 4)

β-Actin:

Forward primer: 5'-TGACAGGATGCAGAAGGAGATTAC-3' (SEQ ID NO: 5)

Reverse primer: 5'-TAAAACGCAGCTCAGTAACAGTC-3' (SEQ ID NO: 6)

The PCR products were separated by electrophoresis on a 1.5% agarose gel, and the bands were confirmed using the G;BOX EF imaging system (Syngene, Cambridge, UK). β-actin was used as an internal standard.

As a result, as shown in Fig. 1, it can be confirmed that the mRNA expression of MuRF1 and atrogin-1, which are involved in muscle protein degradation, increased by dexamethasone (DEX), whereas the mRNA expression of MuRF1 and atrogin-1 decreased when treated with the water extract of Example 1 and the ethanol extract of Example 2. This means that the water extract and ethanol extract of Melinzo seeds of the present invention have an excellent ability to suppress the expression of biomarkers involved in muscle protein degradation in muscle cells.

Test Example 2: Inhibitory effect of Melinzo seed extract on muscle protein degradation

The test was conducted three times in the same manner as Experimental Example 1, except that C2C12 myoblasts were treated with 80 μg/mL of each of the melinzo seed extracts according to Examples 3 to 7. Next, the band densities of MuRF1 and atrogin-1 mRNA were measured using the Image J program (National Institute of Health, Bethesda, MD, USA), and each measurement value was expressed as the mean ± standard deviation by calculating the percentage (%) relative to the control group not treated with dexamethasone (DEX).

As a result, as shown in Table 1, the mRNA expression levels of MuRF1 and atrogin-1 significantly (##p < 0.01) increased compared to the control group when treated with dexamethasone, but when treated with 80 μg/mL of the melinzo seed extract according to the example, it was confirmed that the mRNA expression levels of both MuRF and Atrogin-1 significantly (**p < 0.01) decreased compared to the dexamethasone treatment group. This means that the melinzo seed extract of the present invention has an excellent ability to inhibit muscle protein degradation in muscle cells.

division MuRF-1 expression level
(% vs. control group) Atrogin-1 expression level
(% vs. control group) Control group 100±24.1 100±10.2 Dexamethasone 920.7±43.9 ^## 679.8±45.5 ^## Example 3 454.2±45.1 ^** 169.3±46.1 ^** Example 4 407.5±38.6 ^** 135.9±36.7 ^** Example 5 437.0±40.1 ^** 148.7±41.6 ^** Example 6 453.5±46.6 ^** 178.2±29.0 ^** Example 7 411.2±50.4 ^** 138.6±38.9 ^**

Test Example 3: Effect of increasing mTOR activity, a biomarker of muscle protein synthesis

It is known that mTOR protein, when phosphorylated and activated, can induce the activation of proteins involved in muscle protein synthesis and muscle mass increase in the PI3K/Akt signaling pathway in muscle cells. Therefore, to confirm the muscle production-inducing activity of Melinzo seed extract, the activity of mTOR was confirmed using an mTOR Sandwich ELISA kit (Cell Signaling Technology, Beverly, MA, USA).

C2C12 myoblasts (ATCC, Manassas, VA, USA) were seeded at 5 × 10 5 cells/mL in a 6-well plate with Dulbecco's modified Eagle's media (DMEM; Hyclone) containing 10% fetal ^bovine serum (FBS; Hyclone, Logan, UT, USA) and cultured for 24 hours. When the cell density reached approximately 80–85%, the medium in the well was removed and replaced with DMEM (Hyclone) containing 2% horse serum (HS; Hyclone), and the cells were further cultured for 4 days to differentiate C2C12 cells into myotubes. Next, the melinzo seed water extract of Example 1 was dissolved in DMEM (Hyclone) at concentrations of 40 μg/mL and 80 μg/mL, treated to the cells, and cultured for 12 hours. After incubation, cells were lysed by treatment with cell lysis buffer. The protein in the obtained cell lysate was quantified at a concentration of 1 mg/mL using Bradford (Bio-Rad Laboratories Inc., Hercules, CA, USA). 50 μL of the cell lysate was dispensed into each microwell to which anti-mTOR antibody was attached and incubated at 37°C for 2 hours. After washing four times with washing buffer, detection antibody was treated and incubated at 37°C for 1 hour, and after washing again four times with washing buffer, secondary antibody conjugated with horseradish peroxidase was added and incubated at 37°C for 30 minutes. Finally, after washing four times with washing buffer, TMB substrate was added to each well and left at 37℃ for 10 minutes and stop buffer was added to stop the TMB reaction. After 2 minutes, absorbance was measured at a wavelength of 450 nm to determine the mTOR level in myotube cells treated with melinzo seed extract. The experiment was repeated three times in total, and each measurement value was expressed as the mean ± standard deviation by calculating the percentage (%) of the control group. Differences between groups were confirmed by Duncan multiple range analysis by one-way analysis of variance using the SPSS25.0 statistical package (SPSS Inc., Chicago, IL, USA). At this time, if the p value was less than 5%, it was considered statistically significant.

As a result, as shown in Fig. 2, it can be confirmed that when C2C12 muscle cells were treated with the melinzo seed water extract of Example 1, the activity of mTOR significantly (*p < 0.01) increased compared to the control group. This means that the melinzo seed water extract of the present invention has an excellent ability to promote muscle protein synthesis in muscle cells.

Test Example 4: Effect of Melinzo seed extract on muscle protein synthesis promotion

mTOR activity was measured in the same manner as in Test Example 3, except that C2C12 myoblasts were treated with 80 μg/mL of each of the melinzo seed extracts according to Examples 2 to 7.

As a result, as shown in Table 2, it can be confirmed that the activity of mTOR significantly (**p < 0.01) increased in all cases when treated with the melinzo seed extract according to the embodiment of the present invention compared to the control group that was not treated with the extract. This means that the melinzo seed extract of the present invention has an excellent ability to promote muscle protein synthesis in muscle cells.

division mTOR activation
(% vs. control group) Control group 100±4.3 Example 2 143.2±4.1 ^** Example 3 133.5±3.6 ^** Example 4 145.6±4.5 ^** Example 5 141.1±3.8 ^** Example 6 139.0±4.0 ^** Example 7 143.0±4.4 ^**

Test Example 5: Effects of Melinzo seed powder and extract on muscle function and muscle mass increase in an immobilization animal model

Seven-week-old male mice (C57BL/6J; DBL, Korea) were purchased as experimental animals and the experiment was conducted. The temperature of all animals was maintained at 23 ± 2℃ and the relative humidity at 55 ± 10% in the breeding room. Before the start of the experiment, a total of 25 mice were randomly divided into 5 groups. After 1 week of acclimatization, anesthesia was induced by intraperitoneal injection of 325 mg/kg tribromoethanol (Sigma-Aldrich). After anesthesia, the gastrocnemius muscle and the right sole of the right hindlimb of the mice in the muscular atrophy group and the sample administration group were damaged with a skin stapler (Unidus, Chungcheongbuk-do, Korea) so that the right hindlimb was immobilized, and this condition was maintained for 1 week. After one week, the staples fixed to the gastrocnemius muscle and the sole were removed, and 1,000 mg/kg of the ground melinzo seed, 500 mg/kg of the melinzo seed water extract of Example 1-1, and 500 mg/kg of the melinzo seed ethanol extract of Example 1-2 were orally administered every day for 7 days. At this time, the normal group and the muscular atrophy group were orally administered saline solution instead of the samples.

After the oral administration period, the muscle strength of the rats was measured using a dynamometer (Panlab, Barcelona, Spain). The rats were pulled by the tail with a constant force until they released the end of the dynamometer, and a total of five consecutive tests were performed for each rat.

After muscle strength measurements, the experimental animals were anesthetized by intraperitoneal injection of 325 mg/kg tribromomethanol (Sigma-Aldrich) and sacrificed by cardiac blood sampling. After confirming that the heartbeat had stopped, the intact tibialis anterior muscle was excised from the right hind limb and its weight was measured.

As a result, as shown in Table 3, muscle strength and muscle weight were significantly ( ^## p < 0.01) decreased in the muscular atrophy group compared to the normal group, but it was confirmed that muscle strength and muscle weight were significantly ( ^** p < 0.01) increased in the group treated with the pulverized and extract of Melinzo seeds compared to the muscular atrophy group. This means that the pulverized and extract of Melinzo seeds of the present invention are excellent in increasing muscle strength and muscle weight decreased due to muscular atrophy.

Experimental group Strength (g) Muscle weight (mg) Normal group 263.7±18.4 60.3±4.4 Muscle atrophy 169.0±12.4 ^## 43.5±3.3 ^## Melinzo seed powder 206.1±15.7** 53.1±4.1** Melinzo seed hot water extract 227.9±20.9** 55.9±4.9** Melinzo seed ethanol extract 240.3±22.6** 56.3±5.1**

Hereinafter, examples of manufacturing food, medicine or cosmetics containing the pulverized melinzo seed and extract according to the present invention as an effective ingredient will be described. However, the present invention is not intended to be limited thereto but is merely intended to be described in detail. Using the pulverized melinzo seed or the melinzo seed extract having excellent effects in preventing and treating muscle diseases or improving muscle function, food, medicine or cosmetic compositions of Manufacturing Examples 1 to 3 were manufactured according to the following compositional ingredients and composition ratios according to a conventional method.

<Manufacturing Example 1> Food

Manufacturing Example 1-1. Health Food

The present invention can be manufactured by mixing 1000 mg of the ground melinzo seed or melinzo seed extract, 70 ug of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 ug of vitamin B12, 10 mg of vitamin C, 10 ug of biotin, 1.7 mg of nicotinamide, 50 ug of folic acid, 0.5 mg of calcium pantothenate, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of monobasic potassium phosphate, 55 mg of dibasic calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride, and the mixing ratio may be arbitrarily modified, and the above ingredients may be mixed according to a conventional health food manufacturing method. Next, granules are manufactured and can be used to manufacture a health food composition according to a conventional method.

Manufacturing Example 1-2. Health Drink

1000 mg of the melinzo seed extract of the present invention, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of plum concentrate, and 1 g of taurine are added with purified water to make a total of 900 mL. The above ingredients are mixed according to a general health beverage manufacturing method, stirred and heated at 85° C. for about 1 hour, and the resulting solution is filtered and placed in a sterilized 2 L container, sealed and sterilized, and then stored in the refrigerator. The resulting solution can then be used to manufacture a health beverage composition.

Manufacturing Example 1-3. Chewing gum

Chewing gum was manufactured by a conventional method by mixing 20 wt% of gum base, 76.9 wt% of sugar, 1 wt% of flavoring, 2 wt% of water, and 0.1 wt% of the melinzo seed extract of the present invention.

Manufacturing Example 1-4. Candy

Candy was manufactured by a conventional method by mixing 60 wt% of sugar, 39.8 wt% of corn syrup, 0.1 wt% of a flavoring agent, and 0.1 wt% of the melinzo seed extract of the present invention.

Manufacturing Example 1-5. Biscuit

A biscuit is prepared by mixing 25.59 wt% of grade 1 strength, 22.22 wt% of grade 1 gravity, 4.80 wt% of refined sugar, 0.73 wt% of salt, 0.78 wt% of glucose, 11.78 wt% of palm shortening, 1.54 wt% of ammonium, 0.17 wt% of baking soda, 0.16 wt% of sodium bisulfite, 1.45 wt% of rice flour, 0.0001 wt% of vitamin B, 0.04 wt% of milk flavor, 20.6998 wt% of water, 1.16 wt% of whole milk powder, 0.29 wt% of milk substitute, 0.03 wt% of monobasic calcium phosphate, 0.29 wt% of spraying salt and 7.27 wt% of spraying oil with 0.8301 wt% of the crushed melinzo seed or melinzo seed extract of the present invention using a conventional method. was manufactured.

<Manufacturing Example 2> Medicine

Manufacturing Example 2-1. Sanje

50 mg of the melinzo seed extract of the present invention and 2 g of crystalline cellulose were mixed and then filled into an airtight pouch according to a conventional powder manufacturing method to prepare a powder.

Manufacturing Example 2-2. Refining

50 mg of the melinzo seed extract of the present invention, 400 mg of crystalline cellulose, and 5 mg of magnesium stearate were mixed and then compressed into tablets according to a conventional tablet manufacturing method.

Manufacturing Example 2-3. Capsule

30 mg of the melinzo seed extract of the present invention, 100 mg of whey protein, 400 mg of crystalline cellulose, and 6 mg of magnesium stearate were mixed and then filled into a gelatin capsule according to a conventional capsule manufacturing method to produce a capsule.

<Manufacturing Example 3> Cosmetics

Manufacturing Example 3-1. Nutritious Toner (Milk Lotion)

A nutritious toner was prepared by a conventional method using the melinzo seed extract of the present invention in the nutritious toner formulation ratio shown in Table 4 below.

Mixing ingredients Manufacturing Example 3-1 (weight%) Melinzo seed extract 2.0 Squalane 5.0 beeswax 4.0 Polysorbate 60 1.5 Sorbitan sesquioleate 1.5 Liquid paraffin 0.5 Caprylic/Capric Triglyceride 5.0 glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Triethanolamine 0.2 Preservatives, Colorants, Fragrances Appropriate amount Purified water to 100

Manufacturing Example 3-2. Flexible Toner (Skin Lotion)

A softening lotion was prepared using the melinzo seed extract of the present invention in the formulation ratio of Table 5 below, using a conventional method.

Mixing ingredients Manufacturing Example 3-2 (weight%) Melinzo seed extract 2.0 glycerin 3.0 Butylene glycol 2.0 Propylene glycol 2.0 Carboxyvinyl polymer 0.1 PEG 12 nonylphenyl ether 0.2 Polysorbate 80 0.4 Ethanol 10.0 Triethanolamine 0.1 Preservatives, Colorants, Fragrances Appropriate amount Purified water to 100

Manufacturing Example 3-3. Nourishing Cream

A nutritional cream was prepared using the melinzo seed extract of the present invention in the nutritional cream formulation ratio shown in Table 6 according to a conventional method.

Mixing ingredients Manufacturing Example 3-3 (weight%) Melinzo seed extract 2.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 PEG60 hydrogenated castor oil 2.0 Liquid paraffin 10 Squalane 5.0 Caprylic/Capric Triglyceride 5.0 glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 antiseptic Appropriate amount pigment Appropriate amount spices Appropriate amount Purified water to 100

Manufacturing Example 3-4. Massage Cream

A massage cream was prepared by a conventional method using the crushed melinzo seed or melinzo seed extract of the present invention in the massage cream formulation ratio shown in Table 7 below.

Mixing ingredients Manufacturing Example 3-4 (weight%) Melinzo seed powder or Melinzo seed extract 1.0 beeswax 10.0 Polysorbate 60 1.5 PEG 60 hydrogenated castor oil 2.0 Sorbitan sesquioleate 0.8 Liquid paraffin 40.0 Squalane 5.0 Caprylic/Capric Triglyceride 4.0 glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservatives, Colorants, Fragrances Appropriate amount Purified water to 100

Manufacturing Example 3-5. Pack

A pack was prepared by a conventional method using the crushed melinzo seed or melinzo seed extract of the present invention in the pack formulation ratio shown in Table 8 below.

Mixing ingredients Manufacturing Example 3-5 (weight%) Melinzo seed powder or Melinzo seed extract 1.0 polyvinyl alcohol 13.0 Sodium carboxymethyl cellulose 0.2 glycerin 5.0 Allantoin 0.1 Ethanol 6.0 PEG 12 nonylphenyl ether 0.3 Polysorbate 60 0.3 Preservatives, Colorants, Fragrances Appropriate amount Purified water to 100

Manufacturing Example 3-6. Gel

A gel was prepared using the melinzo seed extract of the present invention according to the gel formulation ratio in Table 9 below, using a conventional method.

Mixing ingredients Manufacturing Example 3-6 (weight%) Melinzo seed extract 0.5 Sodium ethylenediamine acetate 0.05 glycerin 5.0 Carboxyvinyl polymer 0.3 Ethanol 5.0 PEG 60 hydrogenated castor oil 0.5 Triethanolamine 0.3 Preservatives, Colorants, Fragrances Appropriate amount Purified water to 100

In the above table, 'appropriate amount' means that an appropriate amount can be added depending on the preference, and 'to 100' means that the weight % remaining after subtracting the sum of the weight % of the remaining mixing ingredients excluding purified water from the total 100 weight % can be used as the weight % of purified water.

Although the present invention has been described with reference to the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. Furthermore, the appended claims include such modifications and variations as fall within the spirit of the present invention.

As described above, the present invention provides a composition containing melinzo seeds, such as melinzo seed powder or melinzo seed extract, for improving, treating or preventing muscle disease, or for improving muscle function. More specifically, the melinzo seeds of the present invention exhibit excellent effects in improving, treating or preventing muscle disease, or in improving muscle function by reducing the mRNA expression levels of MuRF1 and atrogin-1 and increasing the activity of mTOR, thereby improving muscle mass and muscle strength. Therefore, the present invention can provide a composition exhibiting excellent effects in improving, treating or preventing muscle disease, or in improving muscle function, and thus has high industrial applicability.

Claims (8)

A food composition for improving or preventing muscle disease or improving muscle function, containing melinjo seed as an effective ingredient. In paragraph 1,
The above melinzo seeds are the ground product of melinzo seeds,
The above melinzo seed extract is a food composition for improving or preventing muscle disease or improving muscle function, characterized in that it is obtained by extracting melinzo seeds. In paragraph 1,
A food composition for improving or preventing muscle disease or improving muscle function, characterized in that the above melinzo seed extract is obtained by extracting melinzo seeds 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. In the third paragraph,
A food composition for improving or preventing muscle disease or improving muscle function, characterized in that the organic solvent is at least one solvent selected from the group consisting of alcohol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, and petroleum ether having 1 to 6 carbon atoms. In any one of claims 1 to 4,
A food composition for improving or preventing muscle disease or improving muscle function, characterized in that the muscle disease is at least one selected from the group consisting of sarcopenia, muscular atrophy, muscular dystrophy, atony, muscle degeneration, myasthenia, and cachexia. A pharmaceutical composition for treating or preventing muscle disease containing melinzo seeds as an active ingredient. A cosmetic composition containing melinzo seeds as an active ingredient for improving or preventing muscle disease or improving muscle function. A feed additive containing melinzo seeds as an effective ingredient for improving or preventing muscle disease or improving muscle function.