WO2022139544A1 - Composition for preventing or treating muscle diseases containing aronia or houttuynia cordata extract as active ingredient - Google Patents

Composition for preventing or treating muscle diseases containing aronia or houttuynia cordata extract as active ingredient Download PDF

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WO2022139544A1
WO2022139544A1 PCT/KR2021/019821 KR2021019821W WO2022139544A1 WO 2022139544 A1 WO2022139544 A1 WO 2022139544A1 KR 2021019821 W KR2021019821 W KR 2021019821W WO 2022139544 A1 WO2022139544 A1 WO 2022139544A1
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muscle
extract
aronia
pharmaceutical composition
disease
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PCT/KR2021/019821
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French (fr)
Korean (ko)
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김현수
한정순
김시현
김수진
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고려대학교 산학협력단
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Priority claimed from KR1020210108529A external-priority patent/KR20220092346A/en
Priority claimed from KR1020210183654A external-priority patent/KR20220092402A/en
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Publication of WO2022139544A1 publication Critical patent/WO2022139544A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/78—Saururaceae (Lizard's-tail family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system

Definitions

  • the present invention relates to a composition for muscle strengthening, muscle strengthening, muscle regeneration, or prevention or treatment of muscle disease, and the like, containing aronia and/or eosinthia extract as an active ingredient.
  • Muscle loss and muscle weakness are one of the major physical changes caused by aging, and are known to progress in stages from the age of 40.
  • Skeletal muscle is the largest organ accounting for 50% of the total body weight, and sarcopenia significantly affects the quality of life, such as lowering physical ability and increasing the risk of falls and fractures. expected to be active.
  • Registered Patent Publication No. 10-1617590 'Anti-obesity food composition comprising aronia, acai berry and stevia extract' contains 100 parts by weight of acai berry extract, 50-100 parts by weight of aronia extract and 10-20 parts by weight of stevia extract. that is described. Since muscle loss is also related to the activity of adipocytes, it is being studied a lot with anti-obesity compositions.
  • the present invention is to provide the use of the extract of aronia and / or eosinthia extract for preventing and treating muscle diseases, regenerating and strengthening muscles, and increasing muscle mass.
  • One aspect of the present invention provides a pharmaceutical composition for preventing or treating muscle disease containing an aronia extract as an active ingredient.
  • the aronia extract may contain a cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient.
  • the concentration of the aronia extract may be 0.1 mg/ml to 1000 mg/ml.
  • the aronia extract may be to reduce the blood concentration of lactate dehydrogenase (LDH), creatine kinase (CK), or both.
  • LDH lactate dehydrogenase
  • CK creatine kinase
  • the aronia extract may be extracted with one or more solvents selected from the group consisting of water, C 1 to C 4 lower alcohols, and mixed solvents thereof.
  • the present invention provides a pharmaceutical composition for preventing or treating muscle disease, comprising the cyanidin glycoside of Formula 1 or a salt thereof as an active ingredient.
  • the cyanidin glycoside may be derived from Aronia, and in particular may be obtained from an alcohol extract of Aronia.
  • composition of the present invention may inhibit the expression of myostatin in proportion to the concentration.
  • the composition of the present invention may activate an intracellular protein synthesis mechanism including mTOR.
  • the muscle disease may be a muscle disease due to decreased muscle function, muscle loss, muscle atrophy, muscle wasting or muscle degeneration, and the aforementioned muscle dysfunction, muscle reduction, muscle atrophy, muscle wasting and muscle Regression may be due to anticancer drugs (particularly doxorubicin), dexamethasone, and/or nutritional deficiencies.
  • anticancer drugs particularly doxorubicin
  • dexamethasone particularly doxorubicin
  • nutritional deficiencies particularly doxorubicin
  • the muscle disease is atony, muscular atrophy, muscular dystrophy, myasthenia gravis, cachexia, rigid spinesyndrome, amyotrophic lateral sclerosis ( It may be any one or more selected from the group consisting of ALS, amyotrophic lateral sclerosis, rigid spinsesyndrome, Charcot-Marie-Tooth disease, and sarcopenia.
  • the present invention provides a food composition for improving muscle function, containing an aronia extract as an active ingredient.
  • the present invention provides a food composition for improving muscle function, comprising the cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient.
  • improvement of muscle function means improvement of skeletal muscle function, and includes preventing or delaying weakening of muscle function due to muscle loss, muscle atrophy, muscle wasting, or muscle degeneration.
  • the present invention provides a food composition comprising an aronia extract as an active ingredient to increase or decrease muscle mass.
  • the present invention provides a food composition containing the cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient to inhibit muscle mass increase or muscle decrease.
  • the present invention confirms the muscle cell protection and muscle cell increase effect of the eosinth plant extract, and provides a use for strengthening the muscle, strengthening the muscle, protecting the muscle, and preventing or treating muscle disease.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases, containing the extract of Eosangi as an active ingredient.
  • the muscle disease may be a muscle disease caused by decreased muscle function, muscle loss, muscle atrophy, muscle wasting or muscle degeneration, and atony, muscular atrophy, muscular dystrophy (muscular dystrophy), myasthenia gravis, cachexia, rigid spinesyndrome, amyotrophic lateral sclerosis (amyotrophic lateral sclerosis), rigid spinsesyndrome, Charcot-Marie-Tooth disease (Charcot- It may be any one or more selected from the group consisting of Marie-Tooth disease and sarcopenia.
  • the present invention provides a food composition for improving muscle function, comprising the extract of Eosongcho as an active ingredient.
  • the present invention provides a food composition for inhibiting muscle mass increase and / or muscle loss, comprising the extract of Eosangi as an active ingredient.
  • the eoseongcho extract may be extracted from one or more parts selected from the group consisting of leaves, stems, and roots of eoseongcho.
  • the eosinthia extract may be extracted with one or more solvents selected from the group consisting of water, C 1 to C 4 lower alcohols, and mixed solvents thereof.
  • the eoseongcho extract may be extracted for 48 hours to 120 hours by the cold-chim extraction method.
  • the composition may induce protection of skeletal muscle cells and increase in muscle mass, and the increase in muscle mass is achieved by increasing the number of skeletal muscle cells and/or increasing the size of cells. it could be
  • the composition can inhibit the expression of myostatin, which induces protein degradation and ultimately muscle loss in skeletal muscle.
  • Another aspect of the present invention comprises the steps of extracting raw eoseongcho leaf and stem mixture or raw eoseongcho root in lower alcohol at a temperature below room temperature for 48 to 120 hours; diluting the alcohol extract in distilled water at a ratio of 1/100 to 5/100; and pre-treating the diluted alcohol extract with the muscle cell line c2c12 for at least 24 hours; it provides a method for inhibiting muscle loss, including.
  • the Ewoongcho extract, aronia extract, and cyanidin glycosides derived therefrom which have the effect of preventing and treating muscle diseases, improve the function of skeletal muscle, reduce the expression of proteins inducing muscle loss, and promote the generation and growth of muscles.
  • the composition of the present invention is derived from a natural product whose safety has been verified for a long time, and has the advantage of having very few or no side effects.
  • composition of the present invention in addition to the use of the treatment of muscle diseases, inhibits muscle loss, increases muscle mass, and improves muscle function, for pharmaceutical, quasi-drug, cosmetic material, body vitality enhancement, functional bio material And it is expected to be used in various fields such as for functional food materials.
  • 1 is a flowchart of an alcohol extraction process of aronia.
  • Figure 2 is an MTT analysis graph for testing the muscle cell viability according to Experimental Example 2 after 3 hours of pretreatment with aronia extract.
  • FIG 3 is an MTT analysis graph for testing the muscle cell viability according to Experimental Example 2 after 24 hours of pretreatment with an aronia extract.
  • FIG. 4 is a graph comparing the myostatin expression level according to Experimental Example 3 of the present invention.
  • FIG. 6 is a graph showing the results of the hanging time and holding impulse of the animal experiment according to Experimental Example 1 of the present invention.
  • FIG. 11 is a micro-CT photograph and a CT scan result graph of an animal experiment according to Experimental Example 1 of the present invention.
  • FIG. 13 is a myostatin expression analysis graph according to Experimental Example 2 of the present invention.
  • FIG 17 is a western blot photograph of myostatin and beta-actin according to Experimental Example 3 of the present invention (doxorubicin treatment).
  • 20 is a process chart related to the ethanol extraction process of Eoseongcho.
  • Figure 21 is an MTT analysis graph for testing the muscle cell viability according to Experimental Example 1 after pre-treatment with a mixed extract of Eoseongcho leaf and root for 24 hours.
  • 22 is an MTT analysis graph for testing the muscle cell viability according to Experimental Example 1 after 24 hours of pre-treatment with the Eoseongcho root extract.
  • FIG. 23 is an MTT analysis graph for testing the muscle cell viability according to Experimental Example 1 after 24 hours of pretreatment with a powder obtained by lyophilizing the leaves of Eoseongcho.
  • One aspect of the present invention provides a pharmaceutical composition for preventing or treating muscle disease containing an aronia extract as an active ingredient.
  • the food composition may mean that the active ingredient of aronia fruit is extracted and administered for the prevention or treatment of muscle disease, and is distinguished from the blueberry extract because of the concentrated content of the active ingredient.
  • the aronia extract may be an alcohol extraction after freeze-drying the raw aronia fruit, but is not limited thereto.
  • Another aspect of the present invention relates to a composition for inhibiting sarcopenia containing an eosinosa extract as an active ingredient and a method for inhibiting sarcopenia using the same. % or more provides a functional food composition.
  • Eoseongcho extract of the present invention is an extract obtained by pre-treating any one from the group consisting of eoseongcho leaves, stems, mixtures of leaves and stems and roots by a specific method and extraction under specific conditions, and the extract obtained by concentrating and/or diluted under reduced pressure is effective It is characterized in that it contains as an ingredient.
  • the eoseongcho may be raw eoseongcho, or may be powdered after freeze-drying, and when powdered, eoseongcho leaves are freeze-dried. Depending on the pretreatment process, a large difference in physiological activity may be exhibited. Eoseongcho can be used without restrictions, such as cultivated or commercially available ones.
  • the aronia extract or eosinth extract may be in liquid form or powder form, but is not limited thereto.
  • the extract may have a meaning commonly used as a crude extract, and in a broad sense, a fraction obtained by additionally fractionating the extract may be included. That is, the eosincho extract, aronia extract or its fractions include not only those obtained using an extraction solvent, but also fractions obtained by additionally performing a purification process.
  • the fraction is a fraction obtained by passing the extract through a filtration membrane having a constant molecular weight cut-off value, and various purifications additionally performed such as separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity) It may mean a fraction obtained through the method.
  • the aronia extract may contain a cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient.
  • the aronia contains mostly cyanidin components, and in addition, contains a large amount of tannins that produce astringent taste.
  • Aronia tannins have astringent and intestinal action by binding to proteins, and tannins and their decomposition products combine with metal ions and basic compounds to remove harmful heavy metals and mutagenic substances.
  • metal ions and basic compounds combine with metal ions and basic compounds to remove harmful heavy metals and mutagenic substances.
  • nitrogenous wastes accumulate in large amounts, causing uremia and burdening the kidneys.
  • Formula 1 is a cyanidin-3 glycoside, and is an anthocyanin-based pigment component in which cyanidin is combined with glucose.
  • the cyanidin glycoside of Formula 1 may be a component separated and purified from an alcohol-extracted extract of freeze-dried raw aronia fruit.
  • the aronia extract may further include tannins.
  • the tannins have a large molecular weight, and when a large amount of water-soluble tannins are included, they feel astringent, but during alcohol treatment, they undergo some insolubilization due to the action of alcohol dehydrogenase in the tissue to neutralize the astringent taste.
  • gallic acid or erlagic acid can be produced by insolubilization to promote myocyte differentiation.
  • vegetable tannins have a property of binding well to proteins and can create synergy with cyanidin glycosides by inhibiting the action of proteolytic enzymes.
  • the concentration of the aronia extract may be 0.1 mg/ml to 1000 mg/ml.
  • the concentration of the aronia extract is measured by diluting the freeze-dried aronia fruit in distilled water based on the alcohol-extracted stock.
  • the stock may be 10 g/ml to 50 g/ml, preferably 30 g/ml to 40 g/ml.
  • the aronia extract may be to reduce the blood concentration of lactate dehydrogenase (LDH), creatine kinase (CK), or both.
  • LDH lactate dehydrogenase
  • CK creatine kinase
  • lactate anhydrase and creatine kinase are secreted upon muscle damage, and may be indicators of muscle loss.
  • the active ingredient of the aronia extract may be included as long as it is a component capable of preventing or treating muscle disease or improving muscle function.
  • the active ingredient may include preferably polyphenol, anthocyanin, and catechin, and the polyphenol may include delphinidin, cyanidin, petunidine, pelagonidine, pionidine or malvidin, but most preferably may be cyanidin.
  • composition of the present invention may be added to food to inhibit muscle atrophy, increase muscle mass, and activate muscle hypertrophy.
  • composition of the present invention may be added to food to inhibit muscle atrophy, increase muscle mass, and activate muscle hypertrophy.
  • the aronia extract may contain a cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient.
  • the aronia contains mostly cyanidin components, and in addition, contains a large amount of tannins that produce astringent taste.
  • Aronia tannins have astringent and intestinal action by binding to proteins, and tannins and their decomposition products combine with metal ions and basic compounds to remove harmful heavy metals and mutagenic substances.
  • metal ions and basic compounds combine with metal ions and basic compounds to remove harmful heavy metals and mutagenic substances.
  • nitrogenous wastes accumulate in large amounts, causing uremia and burdening the kidneys.
  • Formula 1 is a cyanidin-3 glycoside, and is an anthocyanin-based pigment component in which cyanidin is combined with glucose.
  • the cyanidin glycoside of Formula 1 may be a component separated and purified from an alcohol-extracted extract of freeze-dried raw aronia fruit.
  • the aronia extract may further include tannins.
  • the tannins have a large molecular weight, and when a large amount of water-soluble tannins are included, they feel astringent, but during alcohol treatment, they undergo some insolubilization due to the action of alcohol dehydrogenase in the tissue to neutralize the astringent taste.
  • gallic acid or erlagic acid can be produced by insolubilization to promote myocyte differentiation.
  • vegetable tannins have a property of binding well to proteins and can create synergy with cyanidin glycosides by inhibiting the action of proteolytic enzymes.
  • the concentration of the aronia extract may be 0.1 mg/ml to 1000 mg/ml.
  • the concentration of the aronia extract is measured by diluting the freeze-dried aronia fruit in distilled water based on the alcohol-extracted stock.
  • the stock may be 10 g/ml to 50 g/ml, preferably 30 g/ml to 40 g/ml.
  • the aronia extract may be to reduce the blood concentration of lactate dehydrogenase (LDH), creatine kinase (CK), or both.
  • LDH lactate dehydrogenase
  • CK creatine kinase
  • lactate anhydrase and creatine kinase are secreted upon muscle damage, and may be indicators of muscle loss.
  • the Aronia extract may inhibit the expression of myostatin in proportion to the concentration.
  • the present inventors confirmed that the aronia extract reduces the expression level of myostatin through a luciferase assay. Muscle loss occurs in proportion to the expression level of myostatin.
  • Another aspect of the present invention provides a method for inhibiting muscle loss, comprising the steps of alcohol extraction of the active ingredient of aronia, diluting the extract of aronia, and treating the diluted extract to damaged muscle cells.
  • the alcohol extraction is different from hot water extraction in that the freeze-dried raw aronia fruit is supported in alcohol or the freeze-dried aronia powder is put in a solvent and extracted at room temperature.
  • the step of treating the diluted extract on the damaged muscle cells includes both a pre-treatment step before inducing damage to the muscle cells and a post-treatment step after inducing damage.
  • the pre-treatment refers to immersion in a diluted extract solution or oral administration for 3 to 36 hours before muscle damage, and post-treatment means treatment after muscle damage occurs.
  • the active ingredient may be a cyanidin glycoside of Formula 1 below or a pharmaceutically effective salt thereof.
  • the extraction may be performed in a lower alcohol having 1 to 4 carbon atoms or less.
  • the lower alcohol having 1 to 4 carbon atoms may be methanol or ethanol, preferably ethanol.
  • the muscle cell damage may be induced by dexamethasone, doxorubicin, or a combination thereof.
  • the dexamethasone is a steroid drug and is mainly used in the preparation of a sarcopenia model.
  • dexamethasone was used as a stress factor for generating muscle atrophy by treating C2C12 muscle cells.
  • Muscular atrophy is a decrease in the size of muscle cells, and may mean that the number of myofibrils in the muscle decreases and the mass of skeletal muscle decreases exponentially with time.
  • the doxorubicin is an anticancer drug that is universally used for various types of cancer patients, but it causes damage to muscle tissue as a side effect of overdose, and in particular, it causes DNA damage in mitochondria among major intracellular organs, resulting in sarcopenia induction model cells It can be used in unit experiments.
  • the muscle cells may include the cell line c2c12.
  • the muscle of the muscle cell may include an anterior tibialis anterior (TA), extensor digitorum longus (EDLL), quadriceps (QC), gastrocnemius (GC), etc., preferably may be anterior tibial muscle.
  • TA anterior tibialis anterior
  • EDLL extensor digitorum longus
  • QC quadriceps
  • GC gastrocnemius
  • aronia extract may be different for each type of muscle, but it has the best effect on the anterior tibialis anterior muscle.
  • the cell line c2c12 can most effectively observe the myostatin expression index.
  • the mouse myoblast line, C2C12 was differentiated to form multinuclear myotubes from mononuclear myoblasts, and the effects of treatment with aronia extract together with dexamethasone were investigated.
  • the aronia extract may have a muscle cell increase rate of 10% to 99%.
  • the muscle cell increase rate was tested for cell viability by treating the muscle cells that did not induce damage with the aronia extract and then performing the MTT test. As a result, it means an increase of 10% to 99% compared to the initial muscle cells.
  • the diluted extract by the treatment of the diluted extract, it may have a cell viability of 90% to 120% compared to the total intact muscle cells.
  • the cell viability was tested after the MTT test was performed after treatment of the aronia extract in the damaged muscle cells. As a result, it was confirmed that the cells were recovered to 90% to 120% based on 100% of the original undamaged muscle cells.
  • the cell viability may refer to the extent to which the reduction in diameter of c2c12 cells is recovered by administration of dexamethasone.
  • a decrease in the diameter of the c2c12 cells means a decrease in the thickness of the skeletal muscle cells, and when the survival rate is 90% or more due to good recovery, the decrease in the thickness of the skeletal muscle cells may be 0% or more and less than 10%.
  • the treatment of the diluted extract may be to support the diluted extract on the damaged muscle cells for 12 to 48 hours.
  • the treatment of the diluted extract may be, preferably, pre-treating the diluted extract to the muscle cells before inducing damage, and then supporting the muscle cells again for 12 to 48 hours after the injury.
  • muscle protection or prevention and treatment can be carried out at the same time, and due to the nature of the natural product, it is possible to proceed without side effects.
  • a pharmaceutical composition or food composition for inhibiting muscle loss using a natural product increases the half-life in the body, the frequency of administration can be freely adjusted, and the cost can be reduced, so it has advantages over direct intake of protein or natural amino acids.
  • the method for inhibiting muscle loss preferably, the steps of extracting the active ingredient of freeze-dried and powdered aronia in 80% or more lower alcohol for 48 hours to 96 hours; diluting the alcohol extract to 1/100,000 to 1/1,000 in distilled water; and pre-treating the diluted alcohol extract to the muscle cell line c2c12 for at least 24 hours; may include
  • Another aspect of the present invention provides a pharmaceutical composition comprising the food composition for inhibiting muscle loss according to an embodiment of the present invention.
  • the pharmaceutical composition may include a cyanidin glycoside as an active ingredient.
  • the cyanidin glycoside of the pharmaceutical composition may preferably be a glycoside extracted from Aronia, but is not limited thereto, and any natural product capable of obtaining a high concentration of cyanidin glycoside may be included.
  • the pharmaceutical composition may be used for at least one selected from the group consisting of Cushing's syndrome, steroid myopathy, polymyositis, peripheral neuropathy, cachexia and inflammatory myositis.
  • the pharmaceutical composition may be preferably used for the treatment of Cushing's syndrome, steroid myopathy, polymyositis, and inflammatory myositis, and may be used as an appetite enhancer for cachexia patients.
  • Eoseongcho extract in muscle cell lines could inhibit muscle cell damage and apoptosis caused by dexamethasone, and on the contrary, the survival rate of muscle cells was increased.
  • This is the same principle as the muscle mass increases by increasing the thickness or size of the muscle in the process of repairing the wound with good protein intake and rest after cells are damaged or torn.
  • the functional food composition containing the extract of Eosangi as an active ingredient can be used not only for inhibiting muscle loss, but also for increasing muscle mass, and may further include other auxiliary additives ingested for the purpose of increasing muscle mass.
  • the muscle cells increased or the cell viability was 93% or more, and the muscle cell viability was dexamethasone. It may mean the degree of recovery of the reduction in the diameter of c2c12 cells by administration.
  • a decrease in the diameter of c2c12 cells means a decrease in the thickness of skeletal muscle cells.
  • the viability is greater than 93%, the reduction in skeletal muscle cell thickness may be less than 7%.
  • Another aspect of the present invention comprises the steps of extracting raw eoseongcho leaf and stem mixture or raw eoseongcho root in lower alcohol at a temperature below room temperature for 48 to 120 hours; diluting the alcohol extract in distilled water at a ratio of 1/100 to 5/100; and pre-treating the diluted alcohol extract with the muscle cell line c2c12 for at least 24 hours; it provides a method for inhibiting muscle loss, including.
  • the lower alcohol may be methanol or ethanol, preferably edible ethanol.
  • the alcohol extraction may include alcohol extraction, and preferably at a low temperature below room temperature, when the extraction is performed for 48 to 120 hours, the cell viability of muscle atrophy-induced muscle cells can be most effectively improved.
  • concentration under reduced pressure and dry powdering step may further include.
  • Aronia (Aronia melanocarpa, 2020) was grown and harvested in Deogyusan, Jeollabuk-do.
  • the supernatant was filtered and concentrated for 5 minutes at 80° C. in a vacuum concentrator to obtain 6.5 mL of a liquid extract and used as a sample.
  • the aronia extract stock concentration was 38.952 g/ml.
  • the stock of Preparation Example was diluted in distilled water at a ratio of 1/10,000 to a concentration of 3.895 mg/ml.
  • the stock of Preparation Example was diluted to a concentration of 38.950 mg/ml by diluting it in distilled water at a ratio of 1/1000.
  • the stock of Preparation Example was diluted with distilled water at a ratio of 1/00 to a concentration of 389.50 mg/ml.
  • the stock of Preparation Example was diluted in distilled water at a ratio of 1/1,000,000 to a concentration of 0.038 mg/ml.
  • the stock of Preparation Example was diluted to a concentration of 0.389 mg/ml by diluting it in distilled water at a ratio of 1/100,000.
  • the experiments conducted were grip strength and hanging time & holding impulse tests to observe changes in muscle force, blood analysis to observe muscle damage indicators, weight measurement by separating muscles, and observation of changes in muscle size with micro CT, weight and food intake Observation and ELIZA TEST were performed.
  • the grip strength was decreased in the nerve amputated animals (NTX), and the grip strength increased in the animals administered with the aronia extract, indicating that the aronia extract was used for muscle function ( power) was confirmed to have contributed to the increase.
  • the hanging time and holding impulse were decreased in the ganglion amputated animals, and although not statistically significant in the animals administered with the aronia extract, both indicators increased. Through this, it can be seen that the aronia extract can be involved in the increase in muscle function.
  • TA anterior tibialis anterior
  • EDLL extensor digitorum longus
  • QC quadriceps
  • GC gastrocnemius
  • the muscle weight of TA was decreased in the ganglioned animals, and it was confirmed that the weight of the TA was statistically significantly increased in the animals administered with aronia. This means that the aronia extract was involved in the increase in the size (size) of the TA muscle.
  • the lean mass decreased in the ganglion amputated animals increased in the aronia extract-administered animals, and the increased fat mass decreased in the aronia extract-administered animals. Although not a significant change, it was confirmed that aronia extract could be involved in improving muscle and fat-related functions.
  • the ELISA test was performed to analyze the indicator by observing the myostatin blood concentration.
  • Aronia extract was analyzed and observed after treatment in the skeletal muscle cell line C2C12.
  • the MTT test was performed to analyze the cell viability and recovery rate.
  • aronia extracts of all concentrations showed almost no cytotoxicity to C2C12 cells. Furthermore, it was confirmed that the number of surviving cells increased when the dilution of 1/10,000 or more was treated. Specifically, when the 1/1,000 dilution was treated, the cell viability increased by 16%, and when the 1/100 dilution was treated, the cell viability increased by 80%.
  • C2C12 cell line was pre-treated with the diluted extract of Example 1 for 3 hours, dexamethasone was treated to induce damage, and cell viability was confirmed after 24 hours. The result is shown in the graph of FIG. 2 .
  • C2C12 cell line was pre-treated with the diluted extract of Example 1 for 24 hours, and then treated with dexamethasone to induce damage and check cell viability after 24 hours.
  • the result is the same as the graph of FIG. 3 .
  • Luciferase assay was performed to analyze the expression level of myostatin as an indicator of muscle reduction inhibition by treating the diluted extracts of Examples 1, 4 and 5. Specifically, a C2C12 cell line in which Myostatin luciferase vector (pMSTN_Luc) is overexpressed was used, and after seeding MSTN_Luc-C2C12 cells, the diluted extracts of Examples 1, 4 and 5 were treated for 12 hours the next day. Then, 1X PLB (1X passive lysis buffer) was added to the culture medium to lyse the cells, and the cell lysate was put into a 96-well plate (white plate) for luciferase assay. The activity was measured and quantified using Bradford reagent.
  • pMSTN_Luc Myostatin luciferase vector
  • the blueberry extract was obtained by preparing a blueberry stock in the same manner as in the preparation of the aronia stock of Preparation Example 1, and diluting the stock in distilled water at a ratio of 1/100,000.
  • blueberry extract also suppressed myostatin expression in skeletal muscle cells, but aronia extract inhibited myostatin expression at a higher level than blueberry (* p-value ⁇ 0.05, ** p-value ⁇ 0.01).
  • Cyanidin the main active ingredient of the Aronia extract, was analyzed and observed after treatment in the skeletal muscle cell line C2C12.
  • Cyanidin was treated with 0.2 ug/ml, 2 ug/ml, and PCR was performed, and then, myostatin mRNA was observed.
  • the muscle reduction inhibitory effect of Aronia is due to inhibition of myostatin expression of cyanidin, the main component, and promotion of cell growth and protein synthesis mechanism, and by confirming that it inhibits increase in myostatin expression caused by dexamethasone and doxorubicin. It can be utilized for suppressing Cushing's syndrome and cancer cachexia by using anticancer drugs.
  • the present invention relates to a composition for inhibiting muscle loss containing an aronia extract as an active ingredient, wherein the aronia extract is a functional food composition having a muscle cell survival rate of 90% or more when administered together with dexamethasone to a muscle cell c2c12 cell line induced to differentiate. to provide (cell viability analysis experiment of Experimental Example 2).
  • cyanidin the main component of Aronia, suppressed the expression level of myostatin, a target protein for controlling muscle mass. It was confirmed that cyanidin inhibited the increase in myostatin protein expression caused by dexamethasone and the anticancer drug doxorubicin (Western blot of Experimental Example 3).
  • the alcohol extraction process extracted in ethanol at room temperature for a long time by using the alcohol extraction process extracted in ethanol at room temperature for a long time, the half-life in the body can be increased, the frequency of administration can be controlled, and the effect of inhibiting muscle loss with few pharmacological side effects can be expected.
  • it can be used for the prevention and treatment of Cushing's syndrome caused by long-term use of dexamethasone, and cachexia caused by anticancer drugs such as doxorubicin.
  • Eoseongcho is refrigerated in 750 mL of 80% ethanol for 96 hours and filtered through filter paper. After centrifugation at 9,000 rpm in a centrifuge for 15 minutes at 4°C, the supernatant was filtered and concentrated at 80°C in a vacuum concentrator for 5 minutes to obtain 5 mL of a liquid extract and used as a sample.
  • the extract obtained as a sample was diluted in distilled water at a ratio of 3/100 and used as an active ingredient in a functional food composition.
  • the muscle cell line C2C12 was pre-treated with the diluted eoseongcho extract obtained as an active ingredient in the above Preparation Example for 24 hours.
  • eoseongcho extract diluted in the muscle cell line C2C12 and 30 uM of Dexamethasone were simultaneously treated again for 24 hours to induce sarcopenia.
  • the muscle cell line C2C12 was diluted with distilled water to a concentration of 3/100 and pre-treated for 24 hours. nephropathy was induced.
  • the muscle cell line C2C12 was treated with dexamethasone to induce apoptosis of muscle cells, and the change in muscle cell viability according to the pretreatment of the Echo extract was confirmed by performing MTT assay.
  • both the dilute eoseongcho extract and the diluted eoseongcho powder have a muscle reduction inhibitory effect, and when treated alone without inducing muscle atrophy, it was confirmed that the amount of muscle increased.
  • each eoseongcho extract was prepared by mixing the eoseongcho extract of Preparation Example and the dried eoseongcho leaf dry powder extract of Example 3 with distilled water and diluting 1/100.
  • the C2C12 cell line overexpressing the Myostatin luciferase vector (pMSTN_Luc) was seeded and treated with each Ewoongcho extract prepared in advance the next day.
  • 1X PLB (1X passive lysis buffer) was added to the culture medium to lyse the cells, and then the cell lysate was put in a 96-well plate (white plate) dedicated to luciferase assay, and LAR II reagent was added, and the plate was then machined (Multiplate enspire). ) to measure the activity and quantified using Bradford reagent.
  • the present invention relates to a composition having the effect of protecting muscle cells, improving muscle function, and increasing muscle mass, and can be used in pharmaceuticals and quasi-drugs for the prevention and treatment of muscle diseases using ingredients derived from natural products, improving muscle function and increasing muscle mass It can be used as food, health functional food, cosmetic material, etc.

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Abstract

The present invention relates to a food composition for suppressing sarcopenia, containing an aronia extract or a Houttuynia cordata extract as an active ingredient, and provides a composition containing cyanidin glycoside or a pharmaceutically effective salt thereof. According to the present invention, aronia-derived cyanidin and a Houttuynia cordata extract prevent muscle loss by inhibiting the expression level of myostatin, and a pharmacological effect with excellent safety can be expected through a natural product extraction process.

Description

아로니아 또는 어성초 추출물을 유효성분으로 함유하는 근육질환 예방 또는 치료용 조성물Composition for the prevention or treatment of muscle disease containing aronia or eosinthia extract as an active ingredient
본 발명은 아로니아 및/또는 어성초 추출물을 유효성분으로 함유하는 근력강화, 근육증강, 근육재생, 또는 근육질환 예방 또는 치료용 조성물 등에 관한 것이다.The present invention relates to a composition for muscle strengthening, muscle strengthening, muscle regeneration, or prevention or treatment of muscle disease, and the like, containing aronia and/or eosinthia extract as an active ingredient.
최근 고령인구가 전세계적으로 급증하는 추세에 있는 가운데, UN에 따르면 2050년에는 60세 이상 인구가 20억명이 넘을 것으로 예측되고 있다. 근육 감소와 근력 약화는 노화로 인해 일어나는 주요 신체 변화증상 중 하나로, 40세 이상부터 단계적으로 진행되는 것으로 알려져 있다. 골격근은 전체 몸무게의 50%에 달하는 가장 큰 기관이며, 근감소증(sarcopenia)은 신체능력을 저하시키고 낙상과 골절의 위험을 높이는 등 삶의 질에 큰 영향을 미치기 때문에 근감소증에 대한 연구는 앞으로 더 활발해질 것으로 예상된다. According to the United Nations, the number of people aged 60 and over is expected to exceed 2 billion by 2050 while the aging population is rapidly increasing worldwide. Muscle loss and muscle weakness are one of the major physical changes caused by aging, and are known to progress in stages from the age of 40. Skeletal muscle is the largest organ accounting for 50% of the total body weight, and sarcopenia significantly affects the quality of life, such as lowering physical ability and increasing the risk of falls and fractures. expected to be active.
근감소의 치료법으로는 미토콘드리아 생성 증가, 근육 단백질 분해억제, 항염제 등이 제시되고 있으나 뚜렷한 치료약이 없는 실정이다. 근감소 치료의 일환으로, 호르몬 치료가 대표적으로 행해지고 있으나 그다지 효과적이지 않고 부작용들이 속속들이 보고되고 있으며, 끼니마다 25 내지 30 g에 해당하는 양질의 단백질을 섭취하는 식이요법도 제안되었으나, 이는 달걀 4 내지 5개 또는 닭가슴살 약 120 g을 매 끼니마다 섭취해야만 도달할 수 있는 양으로서 현실적으로 일반인이 실천하기는 매우 어렵다.As a treatment for muscle loss, increased mitochondrial production, inhibition of muscle protein breakdown, and anti-inflammatory drugs have been suggested, but there is no clear therapeutic agent. As part of the treatment for muscle loss, hormone therapy is typically performed, but it is not very effective and side effects are reported one after another. It is an amount that can only be reached by eating 5 chicken breasts or about 120 g of chicken breast at every meal, and it is very difficult for ordinary people to practice in reality.
따라서 최근 많은 사람들이 단백질 보충제를 대안으로 선택하고 있으나, 단백질 보충제의 섭취는 단백질 과다 섭취의 원인이 될 수 있어 부작용이 발생할 가능성이 높다. 더욱이 신장 질환이 있는 경우에는 고단백질 식이를 수행할 수 없고, 노화가 진행됨에 따라 신장의 기능 또한 감소하므로 근감소증의 예방을 위해서 고단백질 섭취 이외의 다른 대안이 필요하다.Therefore, recently, many people are choosing protein supplements as an alternative, but the intake of protein supplements can cause excessive protein intake, which is highly likely to cause side effects. Moreover, if you have kidney disease, you cannot perform a high-protein diet, and as the aging progresses, the kidney function also decreases.
등록특허공보 10-1617590호 '아로니아, 아사이베리 및 스테비아 추출물을 포함하는 항비만 식품 조성물'에는 아사이베리 추출물 100 중량부, 아로니아 추출물 50~100 중량부 및 스테비아 추출물 10~20 중량부를 포함하는 것이 기재되어 있다. 근감소는 지방세포의 활성과도 연관이 있어, 비만 예방 조성물과 함께 많이 연구되고 있고, 이에 착안하여 아로니아의 유효 성분을 추출하여 근감소 억제를 위한 마이오스타틴 활성 억제 용도를 연구하였다.Registered Patent Publication No. 10-1617590 'Anti-obesity food composition comprising aronia, acai berry and stevia extract' contains 100 parts by weight of acai berry extract, 50-100 parts by weight of aronia extract and 10-20 parts by weight of stevia extract. that is described. Since muscle loss is also related to the activity of adipocytes, it is being studied a lot with anti-obesity compositions.
등록특허공보 10-1461098호 '구기자 추출물, 어성초 추출물 및 삼백초 추출물을 유효성분으로 포함하는 비만관련질환의 예방 또는 치료용 약학적 조성물, 식품 조성물'에는 어성초 추출물을 비만관련질환 예방 조성물로 포함하는 것이 기재되어 있다. 근감소는 지방세포의 활성과도 연관이 있어, 비만 예방 조성물과 함께 많이 연구되고 있고, 이에 착안하여 어성초의 유효 성분을 추출하여 근감소 억제를 위한 마이오스타틴 활성 억제 용도를 연구하였다.Registered Patent Publication No. 10-1461098, 'Pharmaceutical composition for the prevention or treatment of obesity-related diseases, food composition containing goji berry extract, eoseongcho extract and ginseng extract as an active ingredient' is described. Since muscle loss is also related to the activity of adipocytes, it is being studied a lot with anti-obesity compositions.
본 발명은 아로니아 및/또는 어성초 추출물의 근육 질환 예방 및 치료, 근육 재생 및 근육 강화, 및 근육 양 증가의 용도를 제공하는 것이다.The present invention is to provide the use of the extract of aronia and / or eosinthia extract for preventing and treating muscle diseases, regenerating and strengthening muscles, and increasing muscle mass.
그러나, 본 발명이 해결하고자 하는 과제는 이상에서 언급한 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 해당 기술분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description.
본 발명의 일 측면은, 아로니아 추출물을 유효성분으로 함유하는 근육 질환 예방 또는 치료용 약학적 조성물을 제공한다.One aspect of the present invention provides a pharmaceutical composition for preventing or treating muscle disease containing an aronia extract as an active ingredient.
일 실시형태에 따를 때, 상기 아로니아 추출물은 하기 화학식 1의 시아니딘 배당체 또는 그 약학적으로 유효한 염을 유효성분으로 함유하는 것일 수 있다.According to one embodiment, the aronia extract may contain a cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient.
Figure PCTKR2021019821-appb-img-000001
Figure PCTKR2021019821-appb-img-000001
일 실시형태에 따를 때, 상기 아로니아 추출물의 농도는 0.1 mg/ml 내지 1000 mg/ml일 수 있다.According to one embodiment, the concentration of the aronia extract may be 0.1 mg/ml to 1000 mg/ml.
일 실시형태에 따를 때, 상기 아로니아 추출물은 젖산탈수소효소(LDH), 크레아틴키나제(CK) 또는 이 둘 모두의 혈중 농도를 감소시키는 것일 수 있다.According to one embodiment, the aronia extract may be to reduce the blood concentration of lactate dehydrogenase (LDH), creatine kinase (CK), or both.
일 실시형태에 따를 때, 상기 아로니아 추출물은 물, C1내지 C4의 저급알코올 및 이들의 혼합용매로 이루어진 군으로부터 선택된 하나 이상의 용매로 추출된 것일 수 있다. According to an embodiment, the aronia extract may be extracted with one or more solvents selected from the group consisting of water, C 1 to C 4 lower alcohols, and mixed solvents thereof.
또한, 본 발명은 상기 화학식 1의 시아니딘 배당체 또는 그의 염을 유효성분으로 포함하는, 근육 질환 예방 또는 치료용 약학적 조성물을 제공한다. 일 실시형태에 따를 때, 상기 시아니딘 배당체는 아로이나 유래의 것일 수 있으며, 특히 아로니아의 알코올 추출물로부터 획득되는 것일 수 있다. In addition, the present invention provides a pharmaceutical composition for preventing or treating muscle disease, comprising the cyanidin glycoside of Formula 1 or a salt thereof as an active ingredient. According to one embodiment, the cyanidin glycoside may be derived from Aronia, and in particular may be obtained from an alcohol extract of Aronia.
일 실시형태에 따를 때, 본 발명의 조성물은 농도에 비례하여 마이오스타틴의 발현을 억제하는 것일 수 있다.According to one embodiment, the composition of the present invention may inhibit the expression of myostatin in proportion to the concentration.
일 실시형태에 따를 때, 본 발명의 조성물은 mTOR를 포함하는 세포 내 단백질 합성 기전을 활성화시키는 것일 수 있다. According to one embodiment, the composition of the present invention may activate an intracellular protein synthesis mechanism including mTOR.
일 실시형태에 따를 때, 상기 근육 질환은 근 기능 저하, 근육 감소, 근육 위축, 근육 소모 또는 근육 퇴화로 인한 근육 질환일 수 있으며, 상술한 근 기능 저하, 근육 감소, 근육 위축, 근육 소모 및 근육 퇴화는 항암제(특히 독소루비신), 덱사메타손, 및/또는 영양결핍에 의한 것일 수 있다.According to one embodiment, the muscle disease may be a muscle disease due to decreased muscle function, muscle loss, muscle atrophy, muscle wasting or muscle degeneration, and the aforementioned muscle dysfunction, muscle reduction, muscle atrophy, muscle wasting and muscle Regression may be due to anticancer drugs (particularly doxorubicin), dexamethasone, and/or nutritional deficiencies.
일 실시형태에 따를 때, 상기 근육 질환은 긴장감퇴증(atony), 근위축증(muscular atrophy), 근이영양증(muscular dystrophy), 근무력증, 악액질(cachexia), 경직성 척추 증후군(rigid spinesyndrome), 근위축성 측삭경화증(루게릭병, amyotrophic lateral sclerosis), 경직성 척추 증후군(rigid spinsesyndrome), 샤르코-마리-투스병 (Charcot-Marie-Tooth disease) 및 근육 감소증(sarcopenia)으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다. According to one embodiment, the muscle disease is atony, muscular atrophy, muscular dystrophy, myasthenia gravis, cachexia, rigid spinesyndrome, amyotrophic lateral sclerosis ( It may be any one or more selected from the group consisting of ALS, amyotrophic lateral sclerosis, rigid spinsesyndrome, Charcot-Marie-Tooth disease, and sarcopenia.
또한, 본 발명은 아로니아 추출물을 유효성분으로 함유하는, 근 기능 개선용 식품 조성물을 제공한다. In addition, the present invention provides a food composition for improving muscle function, containing an aronia extract as an active ingredient.
또한, 본 발명은 상기 화학식 1의 시아니딘 배당체 또는 이의 약학적으로 유효한 염을 유효성분으로 함유하는, 근 기능 개선용 식품 조성물을 제공한다. In addition, the present invention provides a food composition for improving muscle function, comprising the cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient.
일 실시형태에 따를 때, 근 기능의 개선이란 골격근 기능의 개선을 의미하며, 근육 감소, 근육 위축, 근육 소모 또는 근육 퇴화로 인한 근 기능 약화를 예방하거나 이를 지연시키는 것을 포함하는 의미이다.According to one embodiment, improvement of muscle function means improvement of skeletal muscle function, and includes preventing or delaying weakening of muscle function due to muscle loss, muscle atrophy, muscle wasting, or muscle degeneration.
또한, 본 발명은 아로니아 추출물을 유효성분으로 포함하는 근육 양(muscle mass) 증가 또는 근육 감소 억제 식품 조성물을 제공한다. In addition, the present invention provides a food composition comprising an aronia extract as an active ingredient to increase or decrease muscle mass.
또한, 본 발명은 상기 화학식 1의 시아니딘 배당체 또는 이의 약학적으로 유효한 염을 유효성분으로 함유하는 근육 양(muscle mass) 증가 또는 근육 감소 억제 식품 조성물을 제공한다.In addition, the present invention provides a food composition containing the cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient to inhibit muscle mass increase or muscle decrease.
본 발명은 어성초 추출물의 근육세포 보호 및 근육세포 증가 효과를 확인하여 이를 근력강화, 근육증강, 근육보호, 및 근육질환 예방 또는 치료 용도를 제공하는 것이다.The present invention confirms the muscle cell protection and muscle cell increase effect of the eosinth plant extract, and provides a use for strengthening the muscle, strengthening the muscle, protecting the muscle, and preventing or treating muscle disease.
그러나, 본 발명이 해결하고자 하는 과제는 이상에서 언급한 과제로 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 해당 기술분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those of ordinary skill in the art from the following description.
본 발명은 어성초 추출물을 유효성분으로 함유하는 근육 질환 예방 또는 치료용 약학적 조성물을 제공한다. The present invention provides a pharmaceutical composition for the prevention or treatment of muscle diseases, containing the extract of Eosangi as an active ingredient.
본 발명의 일 예에 따르면, 상기 근육 질환은 근 기능 저하, 근육 감소, 근육 위축, 근육 소모 또는 근육 퇴화로 인한 근육 질환인 것일 수 있으며, 긴장감퇴증(atony), 근위축증(muscular atrophy), 근이영양증(muscular dystrophy), 근무력증, 악액질(cachexia), 경직성 척추 증후군(rigid spinesyndrome), 근위축성 측삭경화증(루게릭병, amyotrophic lateral sclerosis), 경직성 척추 증후군(rigid spinsesyndrome), 샤르코-마리-투스병 (Charcot-Marie-Tooth disease) 및 근육 감소증(sarcopenia)으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있다. According to one embodiment of the present invention, the muscle disease may be a muscle disease caused by decreased muscle function, muscle loss, muscle atrophy, muscle wasting or muscle degeneration, and atony, muscular atrophy, muscular dystrophy (muscular dystrophy), myasthenia gravis, cachexia, rigid spinesyndrome, amyotrophic lateral sclerosis (amyotrophic lateral sclerosis), rigid spinsesyndrome, Charcot-Marie-Tooth disease (Charcot- It may be any one or more selected from the group consisting of Marie-Tooth disease and sarcopenia.
또한, 본 발명은 어성초 추출물을 유효성분으로 포함하는 근 기능 개선용 식품 조성물을 제공한다. In addition, the present invention provides a food composition for improving muscle function, comprising the extract of Eosongcho as an active ingredient.
또한, 본 발명은 어성초 추출물을 유효성분으로 포함하는 근육 양(muscle mass) 증가 및/또는 근육 감소 억제용 식품 조성물을 제공한다. In addition, the present invention provides a food composition for inhibiting muscle mass increase and / or muscle loss, comprising the extract of Eosangi as an active ingredient.
본 발명의 일 예에 따르면, 상기 어성초 추출물은 어성초의 잎, 줄기, 및 뿌리로 이루어진 군으로부터 선택된 1종 이상의 부위로부터 추출된 것일 수 있다. According to an example of the present invention, the eoseongcho extract may be extracted from one or more parts selected from the group consisting of leaves, stems, and roots of eoseongcho.
본 발명의 다른 예에 따르면, 상기 어성초 추출물은 물, C1내지 C4의 저급알코올 및 이들의 혼합용매로 이루어진 군으로부터 선택된 하나 이상의 용매로 추출된 것일 수 있다. According to another example of the present invention, the eosinthia extract may be extracted with one or more solvents selected from the group consisting of water, C 1 to C 4 lower alcohols, and mixed solvents thereof.
본 발명의 또 다른 예에 따르면, 상기 어성초 추출물은 냉침 추출 방법으로 48시간 내지 120시간 동안 추출된 것일 수 있다. According to another example of the present invention, the eoseongcho extract may be extracted for 48 hours to 120 hours by the cold-chim extraction method.
본 발명의 또 다른 예에 따르면, 상기 조성물은 골격근 세포의 보호 및 근육 양 증가를 유도하는 것일 수 있으며, 상기 근육 양의 증가는 골격근 세포의 수의 증가 및/또는 세포의 크기의 증가로 달성되는 것일 수 있다. According to another example of the present invention, the composition may induce protection of skeletal muscle cells and increase in muscle mass, and the increase in muscle mass is achieved by increasing the number of skeletal muscle cells and/or increasing the size of cells. it could be
본 발명의 또 다른 예에 따르면, 상기 조성물은 골격근에서 단백질의 분해와 궁극에 근육 소실을 유도하는 마이오스타틴의 발현을 억제할 수 있다. According to another example of the present invention, the composition can inhibit the expression of myostatin, which induces protein degradation and ultimately muscle loss in skeletal muscle.
본 발명의 또 다른 측면은, 생 어성초 잎과 줄기 혼합 또는 생 어성초 뿌리를 상온 이하 온도의 저급 알코올에서 48시간 내지 120시간 냉침하여 추출하는 단계; 상기 알코올 추출물을 증류수에 1/100 내지 5/100 비율로 희석시키는 단계; 및 상기 희석된 알코올 추출물을 근육세포주 c2c12에 24시간 이상 전처리하는 단계;를 포함하는, 근감소 억제 방법을 제공한다.Another aspect of the present invention comprises the steps of extracting raw eoseongcho leaf and stem mixture or raw eoseongcho root in lower alcohol at a temperature below room temperature for 48 to 120 hours; diluting the alcohol extract in distilled water at a ratio of 1/100 to 5/100; and pre-treating the diluted alcohol extract with the muscle cell line c2c12 for at least 24 hours; it provides a method for inhibiting muscle loss, including.
본 발명에 따른 근육 질환 예방 및 치료 효과를 갖는 어성초 추출물, 아로니아 추출물및 이의 유래의 시아니딘 배당체는 골격근의 기능을 향상시키고 근 손실을 유도하는 단백질의 발현을 감소시키며 근육의 발생 및 성장을 증진하는 신호전달 경로를 활성화시키는바, 근육 질환의 개선, 예방, 및 치료 효과를 갖는다. 또한, 본 발명의 조성물은 오랫동안 높은 안전성이 검증된 천연물 유래의 것으로서 사용에 따른 부작용이 매우 적거나 없다는 장점을 가진다. 이에, 본 발명의 조성물은 근육 질환의 치료 등의 용도 외에도 근 손실을 억제하고 근량을 증가시키며 근육의 기능을 향상시키기 위한 의약용, 의약부외용, 향장소재용, 신체 활력 증진, 기능성 바이오 소재용 및 기능성 식품소재용 등 다양한 방면으로 이용될 것으로 기대된다.According to the present invention, the Ewoongcho extract, aronia extract, and cyanidin glycosides derived therefrom, which have the effect of preventing and treating muscle diseases, improve the function of skeletal muscle, reduce the expression of proteins inducing muscle loss, and promote the generation and growth of muscles. By activating the signaling pathway of In addition, the composition of the present invention is derived from a natural product whose safety has been verified for a long time, and has the advantage of having very few or no side effects. Accordingly, the composition of the present invention, in addition to the use of the treatment of muscle diseases, inhibits muscle loss, increases muscle mass, and improves muscle function, for pharmaceutical, quasi-drug, cosmetic material, body vitality enhancement, functional bio material And it is expected to be used in various fields such as for functional food materials.
도 1 은 아로니아의 주정 추출 공정에 관한 공정도이다.1 is a flowchart of an alcohol extraction process of aronia.
도 2는 아로니아 추출물을 3시간 전처리한 후, 실험예2에 따른 근육 세포 생존률을 테스트하기 위한 MTT 분석 그래프이다.Figure 2 is an MTT analysis graph for testing the muscle cell viability according to Experimental Example 2 after 3 hours of pretreatment with aronia extract.
도 3은 아로니아 추출물을 24시간 전처리한 후, 실험예2에 따른 근육 세포 생존률을 테스트하기 위한 MTT 분석 그래프이다.3 is an MTT analysis graph for testing the muscle cell viability according to Experimental Example 2 after 24 hours of pretreatment with an aronia extract.
도 4는 본 발명의 실험예3에 따른 마이오스타틴 발현 정도를 비교한 그래프이다.4 is a graph comparing the myostatin expression level according to Experimental Example 3 of the present invention.
도 5는 본 발명의 실험예1에 따른 동물실험의 Grip strength 결과 그래프이다.5 is a graph showing the results of the grip strength of the animal experiment according to Experimental Example 1 of the present invention.
도 6은 본 발명의 실험예1에 따른 동물실험의 Hanging time 과 Holding impulse에 대한 결과 그래프이다.6 is a graph showing the results of the hanging time and holding impulse of the animal experiment according to Experimental Example 1 of the present invention.
도 7은 본 발명의 실험예1에 따른 동물실험의 근육 조직 무게 측정과 골격근 근육량의 사진 비교이다.7 is a photograph comparison of muscle tissue weight measurement and skeletal muscle muscle mass in an animal experiment according to Experimental Example 1 of the present invention.
도 8은 본 발명의 실험예1에 따른 동물실험의 Lean mass 및 fat mass 측정 결과이다.8 is a measurement result of lean mass and fat mass of an animal experiment according to Experimental Example 1 of the present invention.
도 9는 본 발명의 실험예1에 따른 동물실험의 체중변화 및 섭식량 관찰 그래프이다.9 is an observation graph of body weight change and feeding amount of an animal experiment according to Experimental Example 1 of the present invention.
도 10은 본 발명의 실험예 1에 따른 동물실험의 마이오스타틴 혈중 농도 그래프이다.10 is a graph of myostatin blood concentration in an animal experiment according to Experimental Example 1 of the present invention.
도 11은 본 발명의 실험예1에 따른 동물실험의 마이크로 CT 사진과 CT 검사결과 그래프이다. 11 is a micro-CT photograph and a CT scan result graph of an animal experiment according to Experimental Example 1 of the present invention.
도 12는 본 발명의 실험예2에 따른 세포실험에서 세포 손상 없이 처리한 MTT 결과 그래프이다.12 is a graph showing the results of MTT treatment without cell damage in the cell experiment according to Experimental Example 2 of the present invention.
도 13은 본 발명의 실험예2에 따른 마이오스타틴 발현 분석 그래프이다.13 is a myostatin expression analysis graph according to Experimental Example 2 of the present invention.
도 14는 본 발명의 실험예3에 따른 시아니딘의 MTT 결과에 따른 결과 그래프이다.14 is a graph of the results according to the MTT results of cyanidin according to Experimental Example 3 of the present invention.
도 15는 본 발명의 실험예3에 따른 시아니딘의 마이오스타틴 프로모터 활성 그래프이다.15 is a graph of the myostatin promoter activity of cyanidin according to Experimental Example 3 of the present invention.
도 16은 본 발명의 실험예3에 따른 웨스턴 블롯 사진이다.16 is a western blot photograph according to Experimental Example 3 of the present invention.
도 17은 본 발명의 실험예3에 따른 마이오스타틴과 베타 액틴 웨스턴블롯 사진이다(독소루비신 처리).17 is a western blot photograph of myostatin and beta-actin according to Experimental Example 3 of the present invention (doxorubicin treatment).
도 18은 본 발명의 실험예3에 따른 마이오스타틴과 베타 액틴 웨스턴블롯 사진이다(덱사메타손 처리).18 is a western blot photograph of myostatin and beta actin according to Experimental Example 3 of the present invention (dexamethasone treatment).
도 19는 아로니아 추출물과 블루베리 추출물의 마이오스타틴 발현 억제 효능을 비교확인한 결과이다.19 is a result of comparing and confirming the myostatin expression inhibitory effect of aronia extract and blueberry extract.
도 20은 어성초의 에탄올 추출 공정에 관한 공정도이다.20 is a process chart related to the ethanol extraction process of Eoseongcho.
도 21는 어성초 잎과 뿌리 혼합 추출물을 24시간 전처리한 후, 실험예1에 따른 근육 세포 생존률을 테스트하기 위한 MTT 분석 그래프이다.Figure 21 is an MTT analysis graph for testing the muscle cell viability according to Experimental Example 1 after pre-treatment with a mixed extract of Eoseongcho leaf and root for 24 hours.
도 22은 어성초 뿌리 추출물을 24시간 전처리한 후, 실험예1에 따른 근육 세포 생존률을 테스트하기 위한 MTT 분석 그래프이다.22 is an MTT analysis graph for testing the muscle cell viability according to Experimental Example 1 after 24 hours of pre-treatment with the Eoseongcho root extract.
도 23는 어성초 잎을 동결건조한 분말을 24시간 전처리한 후, 실험예1에 따른 근육 세포 생존률을 테스트하기 위한 MTT 분석 그래프이다.23 is an MTT analysis graph for testing the muscle cell viability according to Experimental Example 1 after 24 hours of pretreatment with a powder obtained by lyophilizing the leaves of Eoseongcho.
도 24는 어성초 추출물의 마이오스타틴 발현 억제 효능을 확인한 결과이다.24 is a result of confirming the myostatin expression inhibitory effect of the extract of Eoseongcho.
이하에서, 첨부된 도면을 참조하여 실시예들을 상세하게 설명한다. 그러나, 실시예들에는 다양한 변경이 가해질 수 있어서 특허출원의 권리 범위가 이러한 실시예들에 의해 제한되거나 한정되는 것은 아니다. 실시예들에 대한 모든 변경, 균등물 내지 대체물이 권리 범위에 포함되는 것으로 이해되어야 한다.Hereinafter, embodiments will be described in detail with reference to the accompanying drawings. However, since various changes may be made to the embodiments, the scope of the patent application is not limited or limited by these embodiments. It should be understood that all modifications, equivalents and substitutes for the embodiments are included in the scope of the rights.
실시예에서 사용한 용어는 단지 설명을 목적으로 사용된 것으로, 한정하려는 의도로 해석되어서는 안 된다. 단수의 표현은 문맥상 명백하게 다르게 뜻하지 않는 한, 복수의 표현을 포함한다. 본 명세서에서, "포함하다" 또는 "가지다" 등의 용어는 명세서 상에 기재된 특징, 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것이 존재함을 지정하려는 것이지, 하나 또는 그 이상의 다른 특징들이나 숫자, 단계, 동작, 구성요소, 부품 또는 이들을 조합한 것들의 존재 또는 부가 가능성을 미리 배제하지 않는 것으로 이해되어야 한다.Terms used in the examples are used for the purpose of description only, and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to designate that a feature, number, step, operation, component, part, or a combination thereof described in the specification exists, but one or more other features It should be understood that this does not preclude the existence or addition of numbers, steps, operations, components, parts, or combinations thereof.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 실시예가 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥 상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the embodiment belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not
또한, 첨부 도면을 참조하여 설명함에 있어, 도면 부호에 관계없이 동일한 구성 요소는 동일한 참조부호를 부여하고 이에 대한 중복되는 설명은 생략하기로 한다. 실시예를 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 실시예의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다. In addition, in the description with reference to the accompanying drawings, the same components are given the same reference numerals regardless of the reference numerals, and the overlapping description thereof will be omitted. In describing the embodiment, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the embodiment, the detailed description thereof will be omitted.
어느 하나의 실시예에 포함된 구성요소와, 공통적인 기능을 포함하는 구성요소는, 다른 실시예에서 동일한 명칭을 사용하여 설명하기로 한다. 반대되는 기재가 없는 이상, 어느 하나의 실시예에 기재한 설명은 다른 실시예에도 적용될 수 있으며, 중복되는 범위에서 구체적인 설명은 생략하기로 한다.Components included in one embodiment and components having a common function will be described using the same names in other embodiments. Unless otherwise stated, a description described in one embodiment may be applied to another embodiment, and a detailed description in the overlapping range will be omitted.
이하, 본 발명의 조성물에 대하여 실시예 및 도면을 참조하여 구체적으로 설명하도록 한다. 그러나, 본 발명이 이러한 실시예 및 도면에 제한되는 것은 아니다.Hereinafter, the composition of the present invention will be described in detail with reference to Examples and drawings. However, the present invention is not limited to these examples and drawings.
본 발명의 일 측면은, 아로니아 추출물을 유효성분으로 함유하는 근육 질환 예방 또는 치료용 약학적 조성물을 제공한다.One aspect of the present invention provides a pharmaceutical composition for preventing or treating muscle disease containing an aronia extract as an active ingredient.
상기 식품 조성물은 아로니아 열매의 활성성분을 추출하여 근육 질환 예방 또는 치료용으로 투여할 수 있는 것을 의미할 수 있으며, 활성성분의 농축함량 때문에 블루베리 추출물과는 구분된다.The food composition may mean that the active ingredient of aronia fruit is extracted and administered for the prevention or treatment of muscle disease, and is distinguished from the blueberry extract because of the concentrated content of the active ingredient.
상기 아로니아 추출물은 생 아로니아 열매를 동결 건조한 후 알코올 추출한 것일 수 있으나, 이에 제한되지 않는다.The aronia extract may be an alcohol extraction after freeze-drying the raw aronia fruit, but is not limited thereto.
본 발명의 다른 측면은, 어성초 추출물을 유효성분으로 함유하는 근감소 억제 효능 조성물 및 이를 이용한 근감소 억제 방법에 관한 것으로, 근육세포주 c2c12에 덱사메타손과 함께 투여 시 근육세포가 증가하거나, 세포 생존률이 93% 이상인 기능성 식품 조성물을 제공한다.Another aspect of the present invention relates to a composition for inhibiting sarcopenia containing an eosinosa extract as an active ingredient and a method for inhibiting sarcopenia using the same. % or more provides a functional food composition.
본 발명의 어성초 추출물은 어성초 잎, 줄기, 잎과 줄기의 혼합물 및 뿌리로 이루어지는 군으로부터 어느 하나를 특정한 방법으로 전처리하고 특정 조건으로 추출하여 수득한 추출물 및 이를 감압농축 및/또는 희석한 추출물을 유효성분으로 함유하는 것을 특징으로 한다. 상기 어성초는 생 어성초이거나, 동결건조 후 분말화한 것일 수 있으며, 분말화할 경우 어성초 잎을 동결건조한 것이다. 이러한 전처리과정에 따라 생리활성에서 큰 차이를 나타낼 수 있다. 어성초는 재배한 것 또는 시판되는 것 등 제한없이 사용 가능하다.Eoseongcho extract of the present invention is an extract obtained by pre-treating any one from the group consisting of eoseongcho leaves, stems, mixtures of leaves and stems and roots by a specific method and extraction under specific conditions, and the extract obtained by concentrating and/or diluted under reduced pressure is effective It is characterized in that it contains as an ingredient. The eoseongcho may be raw eoseongcho, or may be powdered after freeze-drying, and when powdered, eoseongcho leaves are freeze-dried. Depending on the pretreatment process, a large difference in physiological activity may be exhibited. Eoseongcho can be used without restrictions, such as cultivated or commercially available ones.
상기 아로니아 추출물 또는 어성초 추출물은 액체 형태 또는 분말 형태일 수 있으나, 이에 제한되지 않는다.The aronia extract or eosinth extract may be in liquid form or powder form, but is not limited thereto.
상기 추출물은 조추출물(crude extract)로 통용되는 의미를 가질 수 있으며, 광의적으로는 추출물을 추가적으로 분획 (fractionation)한 분획물도 포함될 수 있다. 즉 상기 어성초 추출물, 아로니아 추출물 또는 이의 분획물은, 추출 용매를 이용하여 얻은 것뿐만 아니라, 정제과정을 추가적으로 진행하여 수득한 분획물도 포함한다. 상기 분획은, 추출물을 일정한 분자량 컷-오프 값을 갖는 여과막을 통과시켜 얻은 분획, 다양한 크로마토그래피 (크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등 추가적으로 실시된 다양한 정제 방법을 통해 얻어진 분획을 의미할 수 있다.The extract may have a meaning commonly used as a crude extract, and in a broad sense, a fraction obtained by additionally fractionating the extract may be included. That is, the eosincho extract, aronia extract or its fractions include not only those obtained using an extraction solvent, but also fractions obtained by additionally performing a purification process. The fraction is a fraction obtained by passing the extract through a filtration membrane having a constant molecular weight cut-off value, and various purifications additionally performed such as separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity) It may mean a fraction obtained through the method.
일 실시형태에 따를 때, 상기 아로니아 추출물은, 하기 화학식 1의 시아니딘 배당체 또는 그 약학적으로 유효한 염을 유효성분으로 함유하는 것일 수 있다.According to an embodiment, the aronia extract may contain a cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient.
Figure PCTKR2021019821-appb-img-000002
Figure PCTKR2021019821-appb-img-000002
상기 아로니아는 베리류의 대표적 항산화 물질로 알려진 블루베리와 달리 시아니딘 성분이 대부분이며 그 외에 떫은 맛을 내는 탄닌을 다량 포함한다.Unlike blueberries, which are known as representative antioxidants of berries, the aronia contains mostly cyanidin components, and in addition, contains a large amount of tannins that produce astringent taste.
아로니아의 탄닌은 단백질과의 결합에 의해 수렴, 정장작용을 가지며, 탄닌과 그 분해산물은 금속이온, 염기성 화합물과 결합하며 유해 중금속 제거와 변이원성물질의 제거한다. 단백질을 과도하게 섭취할 경우 질소성 노폐물을 다량 축적시켜 요독증을 유발하고 신장에 부담을 주는데, 탄닌의 중금속, 질소 흡착으로 인해 이와 같은 부작용을 예방할 수 있다.Aronia tannins have astringent and intestinal action by binding to proteins, and tannins and their decomposition products combine with metal ions and basic compounds to remove harmful heavy metals and mutagenic substances. In case of excessive protein intake, nitrogenous wastes accumulate in large amounts, causing uremia and burdening the kidneys.
상기 화학식 1은 시아니딘-3배당체이며, 안토시아닌계 색소 성분으로 시아니딘이 포도당(glucose)과 결합된 형태이다. Formula 1 is a cyanidin-3 glycoside, and is an anthocyanin-based pigment component in which cyanidin is combined with glucose.
바람직하게는, 상기 화학식 1의 시아니딘 배당체는 동결 건조한 생 아로니아 열매를 알코올 추출한 추출물로부터 분리 정제된 성분일 수 있다.Preferably, the cyanidin glycoside of Formula 1 may be a component separated and purified from an alcohol-extracted extract of freeze-dried raw aronia fruit.
일 실시형태에 따를 때, 상기 아로니아 추출물은, 탄닌을 더 포함하는 것일 수 있다.According to an embodiment, the aronia extract may further include tannins.
상기 탄닌은, 거대 분자량을 가지며, 수용성 탄닌이 다량 포함될 경우 떫은 맛을 느끼게 하지만, 알코올 처리시 조직내 알코올 탈수소 효소의 작용으로 일부 불용화과정을 거치며 떫은 맛을 중화한다. 또한 불용화에 의해 갈산 또는 에라그산을 생성하여 근세포 분화를 촉진할 수 있다. The tannins have a large molecular weight, and when a large amount of water-soluble tannins are included, they feel astringent, but during alcohol treatment, they undergo some insolubilization due to the action of alcohol dehydrogenase in the tissue to neutralize the astringent taste. In addition, gallic acid or erlagic acid can be produced by insolubilization to promote myocyte differentiation.
특히 식물성 탄닌은 단백질과 잘 결합하는 성질을 가지고, 단백질 분해 효소의 작용을 저해하여 시아니딘 배당체와 시너지를 낼 수 있다.In particular, vegetable tannins have a property of binding well to proteins and can create synergy with cyanidin glycosides by inhibiting the action of proteolytic enzymes.
일 실시형태에 따를 때, 상기 아로니아 추출물의 농도는, 0.1 mg/ml 내지 1000 mg/ml일 수 있다.According to one embodiment, the concentration of the aronia extract may be 0.1 mg/ml to 1000 mg/ml.
아로니아 추출물의 농도는, 동결 건조 아로니아 열매를 알코올 추출한 스톡(stock)을 기준으로 증류수에 희석시킨 것을 측정한다. The concentration of the aronia extract is measured by diluting the freeze-dried aronia fruit in distilled water based on the alcohol-extracted stock.
상기 스톡은 10 g/ml 내지 50 g/ml, 바람직하게는 30 g/ml 내지 40 g/ml 일 수 있다.The stock may be 10 g/ml to 50 g/ml, preferably 30 g/ml to 40 g/ml.
상기 범위 미만의 스톡은 근감소 억제용도를 갖는 아로니아의 활성성분을 충분히 추출하지 못할 수 있고, 범위를 초과할 경우, 비용상 이점이 사라질 수 있다.Stocks below the above range may not be able to sufficiently extract the active ingredient of aronia having the purpose of inhibiting muscle loss, and if it exceeds the range, the cost advantage may disappear.
상기 스톡의 1/100000 내지 1/10까지 아로니아 추출물로 사용해도 근감소 억제 효능을 나타낼 수 있다.Even 1/100000 to 1/10 of the stock can be used as an aronia extract to exhibit muscle loss inhibitory efficacy.
일 실시형태에 따를 때, 상기 아로니아 추출물은, 젖산탈수소효소(LDH), 크레아틴키나제(CK) 또는 이 둘 모두의 혈중 농도를 감소시키는 것일 수 있다.According to one embodiment, the aronia extract may be to reduce the blood concentration of lactate dehydrogenase (LDH), creatine kinase (CK), or both.
상기 젖산탈수효소 및 크레아틴키나제는 근육 손상시 분비되는 것으로, 근감소의 지표가 될 수 있다.The lactate anhydrase and creatine kinase are secreted upon muscle damage, and may be indicators of muscle loss.
상기 아로니아 추출물의 유효성분은, 근육질환을 예방 또는 치료하거나, 근기능을 개선시킬 수 있는 성분이라면 모두 포함될 수 있다. 유효성분은, 바람직하게는 폴리페놀, 안토시아닌, 카테킨을 포함할 수 있고, 상기 폴리페놀은 델피니딘, 시아니딘, 페튜니딘, 펠라고니딘, 피오니딘 또는 말비딘를 포함할 수 있으나, 가장 바람직하게는 시아니딘일 수 있다.The active ingredient of the aronia extract may be included as long as it is a component capable of preventing or treating muscle disease or improving muscle function. The active ingredient may include preferably polyphenol, anthocyanin, and catechin, and the polyphenol may include delphinidin, cyanidin, petunidine, pelagonidine, pionidine or malvidin, but most preferably may be cyanidin.
또한, 본 발명의 조성물은 근위축 억제, 근육량 증가, 근 비대 활성 등을 위하여 식품에 첨가될 수 있다.In addition, the composition of the present invention may be added to food to inhibit muscle atrophy, increase muscle mass, and activate muscle hypertrophy.
또한, 본 발명의 조성물은 근위축 억제, 근육량 증가, 근 비대 활성 등을 위하여 식품에 첨가될 수 있다.In addition, the composition of the present invention may be added to food to inhibit muscle atrophy, increase muscle mass, and activate muscle hypertrophy.
일 실시형태에 따를 때, 상기 아로니아 추출물은, 하기 화학식 1의 시아니딘 배당체 또는 그 약학적으로 유효한 염을 유효성분으로 함유하는 것일 수 있다.According to an embodiment, the aronia extract may contain a cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient.
Figure PCTKR2021019821-appb-img-000003
Figure PCTKR2021019821-appb-img-000003
상기 아로니아는 베리류의 대표적 항산화 물질로 알려진 블루베리와 달리 시아니딘 성분이 대부분이며 그 외에 떫은 맛을 내는 탄닌을 다량 포함한다.Unlike blueberries, which are known as representative antioxidants of berries, the aronia contains mostly cyanidin components, and in addition, contains a large amount of tannins that produce astringent taste.
아로니아의 탄닌은 단백질과의 결합에 의해 수렴, 정장작용을 가지며, 탄닌과 그 분해산물은 금속이온, 염기성 화합물과 결합하며 유해 중금속 제거와 변이원성물질의 제거한다. 단백질을 과도하게 섭취할 경우 질소성 노폐물을 다량 축적시켜 요독증을 유발하고 신장에 부담을 주는데, 탄닌의 중금속, 질소 흡착으로 인해 이와 같은 부작용을 예방할 수 있다.Aronia tannins have astringent and intestinal action by binding to proteins, and tannins and their decomposition products combine with metal ions and basic compounds to remove harmful heavy metals and mutagenic substances. In case of excessive protein intake, nitrogenous wastes accumulate in large amounts, causing uremia and burdening the kidneys.
상기 화학식 1은 시아니딘-3배당체이며, 안토시아닌계 색소 성분으로 시아니딘이 포도당(glucose)과 결합된 형태이다. Formula 1 is a cyanidin-3 glycoside, and is an anthocyanin-based pigment component in which cyanidin is combined with glucose.
바람직하게는, 상기 화학식 1의 시아니딘 배당체는 동결 건조한 생 아로니아 열매를 알코올 추출한 추출물로부터 분리 정제된 성분일 수 있다.Preferably, the cyanidin glycoside of Formula 1 may be a component separated and purified from an alcohol-extracted extract of freeze-dried raw aronia fruit.
일 실시형태에 따를 때, 상기 아로니아 추출물은, 탄닌을 더 포함하는 것일 수 있다.According to an embodiment, the aronia extract may further include tannins.
상기 탄닌은, 거대 분자량을 가지며, 수용성 탄닌이 다량 포함될 경우 떫은 맛을 느끼게 하지만, 알코올 처리시 조직내 알코올 탈수소 효소의 작용으로 일부 불용화과정을 거치며 떫은 맛을 중화한다. 또한 불용화에 의해 갈산 또는 에라그산을 생성하여 근세포 분화를 촉진할 수 있다. The tannins have a large molecular weight, and when a large amount of water-soluble tannins are included, they feel astringent, but during alcohol treatment, they undergo some insolubilization due to the action of alcohol dehydrogenase in the tissue to neutralize the astringent taste. In addition, gallic acid or erlagic acid can be produced by insolubilization to promote myocyte differentiation.
특히 식물성 탄닌은 단백질과 잘 결합하는 성질을 가지고, 단백질 분해 효소의 작용을 저해하여 시아니딘 배당체와 시너지를 낼 수 있다.In particular, vegetable tannins have a property of binding well to proteins and can create synergy with cyanidin glycosides by inhibiting the action of proteolytic enzymes.
일 실시형태에 따를 때, 상기 아로니아 추출물의 농도는, 0.1 mg/ml 내지 1000 mg/ml일 수 있다.According to one embodiment, the concentration of the aronia extract may be 0.1 mg/ml to 1000 mg/ml.
아로니아 추출물의 농도는, 동결 건조 아로니아 열매를 알코올 추출한 스톡(stock)을 기준으로 증류수에 희석시킨 것을 측정한다. The concentration of the aronia extract is measured by diluting the freeze-dried aronia fruit in distilled water based on the alcohol-extracted stock.
상기 스톡은 10 g/ml 내지 50 g/ml, 바람직하게는 30 g/ml 내지 40 g/ml 일 수 있다.The stock may be 10 g/ml to 50 g/ml, preferably 30 g/ml to 40 g/ml.
상기 범위 미만의 스톡은 근감소 억제용도를 갖는 아로니아의 활성성분을 충분히 추출하지 못할 수 있고, 범위를 초과할 경우, 비용상 이점이 사라질 수 있다.Stocks below the above range may not be able to sufficiently extract the active ingredient of aronia having the purpose of inhibiting muscle loss, and if it exceeds the range, the cost advantage may disappear.
상기 스톡의 1/100000 내지 1/10까지 아로니아 추출물로 사용해도 근감소 억제 효능을 나타낼 수 있다.Even 1/100000 to 1/10 of the stock can be used as an aronia extract to exhibit muscle loss inhibitory efficacy.
일 실시형태에 따를 때, 상기 아로니아 추출물은, 젖산탈수소효소(LDH), 크레아틴키나제(CK) 또는 이 둘 모두의 혈중 농도를 감소시키는 것일 수 있다.According to one embodiment, the aronia extract may be to reduce the blood concentration of lactate dehydrogenase (LDH), creatine kinase (CK), or both.
상기 젖산탈수효소 및 크레아틴키나제는 근육 손상시 분비되는 것으로, 근감소의 지표가 될 수 있다.The lactate anhydrase and creatine kinase are secreted upon muscle damage, and may be indicators of muscle loss.
일 실시형태에 따를 때, 상기 아로니아 추출물은, 농도에 비례하여 마이오스타틴의 발현을 억제하는 것일 수 있다.According to an embodiment, the Aronia extract may inhibit the expression of myostatin in proportion to the concentration.
본 발명자들은 아로니아 추출물이 상기 마이오스타틴의 발현 수준을 감소시킴을 luciferase assay를 통해 확인하였다. 마이오스타틴은 발현량에 비례하게 근육 손실이 일어난다.The present inventors confirmed that the aronia extract reduces the expression level of myostatin through a luciferase assay. Muscle loss occurs in proportion to the expression level of myostatin.
본 발명의 다른 측면은, 아로니아의 활성성분을 알코올 추출하는 단계, 상기 아로니아 추출물을 희석시키는 단계 및 상기 희석 추출물을 손상 근육세포에 처리하는 단계를 포함하는, 근감소 억제 방법을 제공한다.Another aspect of the present invention provides a method for inhibiting muscle loss, comprising the steps of alcohol extraction of the active ingredient of aronia, diluting the extract of aronia, and treating the diluted extract to damaged muscle cells.
상기 알코올 추출은 동결 건조된 아로니아 생 열매를 알코올에 담지하거나, 동결 건조 아로니아 분물을 용매에 넣어 상온 추출하는 것으로, 열수 추출과 상이하다.The alcohol extraction is different from hot water extraction in that the freeze-dried raw aronia fruit is supported in alcohol or the freeze-dried aronia powder is put in a solvent and extracted at room temperature.
열수 추출의 경우, 높은 온도로 끓이는 과정에서 아로니아의 주요 활성 성분이 일부 소실될 수 있으며, 특히 시아니딘과 시너지를 내는 탄닌 성분이 소실될 수 있다.In the case of hot water extraction, some of the main active ingredients of aronia may be lost in the process of boiling at a high temperature, and in particular, tannins that synergize with cyanidin may be lost.
상기 희석 추출물을 손상 근육세포에 처리하는 단계는, 근육세포의 손상 유도 전에 전처리 단계, 손상을 유도시킨 후의 후처리 단계 모두를 포함한다.The step of treating the diluted extract on the damaged muscle cells includes both a pre-treatment step before inducing damage to the muscle cells and a post-treatment step after inducing damage.
상기 전처리는, 근육의 손상 전 3시간 내지 36시간 동안 희석 추출물 용액에 담지시키거나, 경구 투여하는 것을 의미하며, 후처리는, 근육의 손상이 발생한 이후, 처리하는 것을 의미한다.The pre-treatment refers to immersion in a diluted extract solution or oral administration for 3 to 36 hours before muscle damage, and post-treatment means treatment after muscle damage occurs.
일 실시형태에 따를 때, 상기 활성성분은, 하기 화학식 1의 시아니딘 배당체 또는 그 약학적으로 유효한 염일 수 있다.According to an embodiment, the active ingredient may be a cyanidin glycoside of Formula 1 below or a pharmaceutically effective salt thereof.
Figure PCTKR2021019821-appb-img-000004
Figure PCTKR2021019821-appb-img-000004
일 실시형태에 따를 때, 상기 추출은, 탄소수 1 내지 4 이하의 저급 알코올에서 수행될 수 있다.According to an embodiment, the extraction may be performed in a lower alcohol having 1 to 4 carbon atoms or less.
상기 탄소수 1 내지 4 이하의 저급 알코올은, 메탄올 또는 에탄올 일 수 있으며, 바람직하게는 에탄올일 수 있다.The lower alcohol having 1 to 4 carbon atoms may be methanol or ethanol, preferably ethanol.
메탄올 또는 에탄올에서 알코올 추출할 경우, 주요 활성성분인 시아니딘-3 배당체와 탄닌의 함량뿐만 아니라, 플라보노이드 함량, DPPH 라디컬 소거활성, 슈퍼옥사이드 라디컬 소거활성, 티로시나제 저해활성 모두 열수 추출에 비해 우수하다.In the case of alcohol extraction from methanol or ethanol, not only the content of cyanidin-3 glycoside and tannin, which are the main active ingredients, but also the flavonoid content, DPPH radical scavenging activity, superoxide radical scavenging activity, and tyrosinase inhibitory activity are all superior to hot water extraction do.
일 실시형태에 따를 때, 상기 근육세포의 손상은 덱사메타손, 독소루비신 또는 이 들의 조합에 의해 유도될 수 있다.According to one embodiment, the muscle cell damage may be induced by dexamethasone, doxorubicin, or a combination thereof.
상기 덱사메타손은 스테로이드 약물로 근감소증 모델 제조에 주로 사용된다. 덱사메타손은 본 발명에서는 C2C12 근육 세포에 처치하여, 근위축을 발생시키기 위한 스트레스 인자로 사용되었다. 근위축은 근세포의 크기가 저하된 것으로, 근육의 근원섬유 숫자가 감소하여 골격근 질량이 시간경과에 따라 지수함수적으로 줄어드는 것을 의미할 수 있다.The dexamethasone is a steroid drug and is mainly used in the preparation of a sarcopenia model. In the present invention, dexamethasone was used as a stress factor for generating muscle atrophy by treating C2C12 muscle cells. Muscular atrophy is a decrease in the size of muscle cells, and may mean that the number of myofibrils in the muscle decreases and the mass of skeletal muscle decreases exponentially with time.
상기 독소루비신은, 다양한 종류의 암환자들에게 범용적으로 사용되는 항암제이나, 과복용에 따른 부작용으로 근육 조직의 손상을 일으키며, 특히 세포내 주요기관 중 미토콘드리아 내 DNA 손상을 일으켜 근감소증 유도 모델의 세포 단위 실험에서 사용될 수 있다.The doxorubicin is an anticancer drug that is universally used for various types of cancer patients, but it causes damage to muscle tissue as a side effect of overdose, and in particular, it causes DNA damage in mitochondria among major intracellular organs, resulting in sarcopenia induction model cells It can be used in unit experiments.
일 실시형태에 따를 때, 상기 근육세포는, 세포주 c2c12를 포함할 수 있다.According to one embodiment, the muscle cells may include the cell line c2c12.
상기 근육세포의 근육은, 전방경골근(TA: tibialis anterior), 장지지신근(EDLL: extensor digitorum longus), 대퇴사두근(QC: quadriceps), 비복근(GC: gastrocnemius) 등을 포함할 수 있으며, 바람직하게는 전방경골근일 수 있다.The muscle of the muscle cell may include an anterior tibialis anterior (TA), extensor digitorum longus (EDLL), quadriceps (QC), gastrocnemius (GC), etc., preferably may be anterior tibial muscle.
아로니아 추출물은 근육의 형태별로 미치는 효과가 상이할 수 있는데, 전방경골근에서 가장 좋은 효과를 가진다.The effect of aronia extract may be different for each type of muscle, but it has the best effect on the anterior tibialis anterior muscle.
세포주 c2c12는 마이오스타틴 발현 지표를 가장 효과적으로 관찰할 수 있다.The cell line c2c12 can most effectively observe the myostatin expression index.
마우스 근원세포주인 C2C12를 단핵의 근원세포 (myoblast)에서 다핵의 근관세포 (myotube)를 형성하도록 분화하여 덱사메타손과 함께 아로니아 추출물을 처리하여 효과를 탐색하였다.The mouse myoblast line, C2C12, was differentiated to form multinuclear myotubes from mononuclear myoblasts, and the effects of treatment with aronia extract together with dexamethasone were investigated.
일 실시형태에 따를 때, 상기 아로니아 추출물은, 10 % 내지 99 %의 근육세포 증가율을 가질 수 있다.According to one embodiment, the aronia extract may have a muscle cell increase rate of 10% to 99%.
상기 근육세포 증가율은 손상을 유도하지 않은 근육세포에 아로니아 추출물을 처리한 후 MTT 테스트를 진행하여 세포 생동성을 실험하였다. 그 결과 최초 근육세포 대비 10 % 내지 99 % 증가한 것을 의미한다.The muscle cell increase rate was tested for cell viability by treating the muscle cells that did not induce damage with the aronia extract and then performing the MTT test. As a result, it means an increase of 10% to 99% compared to the initial muscle cells.
일 실시형태에 따를 때, 상기 희석 추출물의 처리에 의해, 손상되지 않은 근육세포 전체 대비 90 % 내지 120 %의 세포 생존율을 가질 수 있다.According to one embodiment, by the treatment of the diluted extract, it may have a cell viability of 90% to 120% compared to the total intact muscle cells.
상기 세포 생존율은 손상이 유도된 근육세포에 아로니아 추출물을 처리한 후 MTT 테스트를 진행한 후, 세포 생존율을 실험하였다. 그 결과 손상되지 않은 최초 근육세포 100 % 기준 90 % 내지 120 %까지 세포가 회복된 것을 확인할 수 있었다.The cell viability was tested after the MTT test was performed after treatment of the aronia extract in the damaged muscle cells. As a result, it was confirmed that the cells were recovered to 90% to 120% based on 100% of the original undamaged muscle cells.
상기 세포 생존율은 덱사메타손 투여에 의한 c2c12세포의 지름(diameter)감소를 리커버리 시키는 정도를 의미할 수 있다. c2c12세포의 지름(diameter)감소는 곧 골격근세포 두께의 감소를 의미하며, 리커버리가 잘 되어 생존률이 90% 이상이 되는 경우, 골격근세포 두께의 감소가 0% 이상 10% 미만일 수 있다.The cell viability may refer to the extent to which the reduction in diameter of c2c12 cells is recovered by administration of dexamethasone. A decrease in the diameter of the c2c12 cells means a decrease in the thickness of the skeletal muscle cells, and when the survival rate is 90% or more due to good recovery, the decrease in the thickness of the skeletal muscle cells may be 0% or more and less than 10%.
일 실시형태에 따를 때, 상기 희석 추출물의 처리는, 손상 근육세포에 희석 추출물을 12시간 내지 48시간 담지하는 것일 수 있다.According to one embodiment, the treatment of the diluted extract may be to support the diluted extract on the damaged muscle cells for 12 to 48 hours.
상기 희석 추출물의 처리는 바람직하게는 손상 유도 전 근육세포에 희석 추출물을 전처리한 후, 손상 이후 근육세포에 다시 12시간 내지 48시간동안 담지하는 것일 수 있다.The treatment of the diluted extract may be, preferably, pre-treating the diluted extract to the muscle cells before inducing damage, and then supporting the muscle cells again for 12 to 48 hours after the injury.
희석 추출물의 전처리 및 후처리를 통해서 근육의 보호 또는 예방과 치료를 동시에 진행할 수 있으며, 천연물 특성상 부작용 없이 진행이 가능하다.Through pre-treatment and post-treatment of the diluted extract, muscle protection or prevention and treatment can be carried out at the same time, and due to the nature of the natural product, it is possible to proceed without side effects.
천연물을 이용한 근감소 억제용 의약 조성물 또는 식품 조성물은, 체내 반감기를 증대시키며, 투여횟수가 자유롭게 조절 가능하고 비용을 절감시킬 수 있어서 단백질의 직접 섭취나 천연 아미노산 대비 장점을 갖는다.A pharmaceutical composition or food composition for inhibiting muscle loss using a natural product increases the half-life in the body, the frequency of administration can be freely adjusted, and the cost can be reduced, so it has advantages over direct intake of protein or natural amino acids.
일 실시형태에 따를 때, 상기 근감소 억제 방법은, 바람직하게는, 동결 건조 및 분말화한 아로니아의 활성성분을 80% 이상 저급 알코올에서 48시간 내지 96시간 추출하는 단계; 상기 알코올 추출물을 증류수에 1/100,000 내지 1/1,000 로 희석하는 단계; 및 상기 희석된 알코올 추출물을 근육세포주 c2c12에 24시간 이상 전처리하는 단계; 를 포함할 수 있다.According to one embodiment, the method for inhibiting muscle loss, preferably, the steps of extracting the active ingredient of freeze-dried and powdered aronia in 80% or more lower alcohol for 48 hours to 96 hours; diluting the alcohol extract to 1/100,000 to 1/1,000 in distilled water; and pre-treating the diluted alcohol extract to the muscle cell line c2c12 for at least 24 hours; may include
본 발명의 다른 측면은, 본 발명의 일 실시형태에 의한 근감소 억제용 식품 조성물을 포함하는 약학 조성물을 제공한다.Another aspect of the present invention provides a pharmaceutical composition comprising the food composition for inhibiting muscle loss according to an embodiment of the present invention.
상기 약학 조성물은, 시아니딘 배당체를 유효성분으로 포함할 수 있다. 상기 약학 조성물의 시아니딘 배당체는, 바람직하게는 아로니아로부터 추출된 배당체일 수 있으나, 이에 한정되지 않으며, 시아니딘 배당체를 고농도로 수득할 수 있는 천연물이라면 모두 포함될 수 있다.The pharmaceutical composition may include a cyanidin glycoside as an active ingredient. The cyanidin glycoside of the pharmaceutical composition may preferably be a glycoside extracted from Aronia, but is not limited thereto, and any natural product capable of obtaining a high concentration of cyanidin glycoside may be included.
일 실시형태에 따를 때, 상기 약학 조성물은, 쿠싱 증후군, 스테로이드 근병증, 다발성 근염, 말초 신경 병증, 악액질 및 염증성 근염으로 이루어지는 군으로부터 선택된 어느 하나 이상의 용도일 수 있다.According to an embodiment, the pharmaceutical composition may be used for at least one selected from the group consisting of Cushing's syndrome, steroid myopathy, polymyositis, peripheral neuropathy, cachexia and inflammatory myositis.
상기 약학 조성물은 바람직하게는 쿠싱 증후군, 스테로이드 근병증, 다발성 근염, 염증성 근염 치료용일 수 있고, 악액질 환자의 식욕 증진제로 사용될 수 있다.The pharmaceutical composition may be preferably used for the treatment of Cushing's syndrome, steroid myopathy, polymyositis, and inflammatory myositis, and may be used as an appetite enhancer for cachexia patients.
어성초 추출물을 근육세포주에 전처리 하는 경우 덱사메타손에 의한 근육세포 손상과 세포사멸을 억제할 수 있으며, 오히려 근육세포의 생존율이 증가하였다. 이는 세포가 상처가 나거나 찢어진 이후, 좋은 단백질 섭취와 휴식으로 상처가 회복되는 과정에서 근육의 두께 또는 크기가 성장하여 근육양이 증가하는 것과 같은 원리이다. 그 결과 어성초 추출물을 유효성분으로 하는 기능성 식품 조성물은, 근감소 억제 용도 뿐만 아니라, 근육양 증가 용도로도 사용될 수 있으며, 근육양 증가 목적으로 섭취하는 다른 보조 첨가제를 더 포함할 수 있다.Pre-treatment of Eoseongcho extract in muscle cell lines could inhibit muscle cell damage and apoptosis caused by dexamethasone, and on the contrary, the survival rate of muscle cells was increased. This is the same principle as the muscle mass increases by increasing the thickness or size of the muscle in the process of repairing the wound with good protein intake and rest after cells are damaged or torn. As a result, the functional food composition containing the extract of Eosangi as an active ingredient can be used not only for inhibiting muscle loss, but also for increasing muscle mass, and may further include other auxiliary additives ingested for the purpose of increasing muscle mass.
어성초 추출물을 유효성분으로 하는 상기 기능성 식품 조성물은, 근육세포주 c2c12에 덱사메타존과 함께 투여시 근육세포가 증가하거나, 세포 생존률이 93% 이상 인 것을 확인할 수 있었으며, 상기 근육세포 생존률은 덱사메타존 투여에 의한 c2c12세포의 지름(diameter)감소를 리커버리 시키는 정도를 의미할 수 있다. c2c12세포의 지름(diameter)감소는 곧 골격근세포 두께의 감소를 의미하며, 리커버리가 잘 되어 생존률이 90% 이상이 되는 경우, 골격근세포 두께의 감소가 0% 이상 10% 미만일 수 있고. 생존률이 93% 이상이 되는 경우, 골격근세포 두께의 감소가 7% 미만일 수 있다. When the functional food composition containing the extract of Eosangi as an active ingredient was administered together with dexamethasone to the muscle cell line c2c12, it was confirmed that the muscle cells increased or the cell viability was 93% or more, and the muscle cell viability was dexamethasone. It may mean the degree of recovery of the reduction in the diameter of c2c12 cells by administration. A decrease in the diameter of c2c12 cells means a decrease in the thickness of skeletal muscle cells. When the viability is greater than 93%, the reduction in skeletal muscle cell thickness may be less than 7%.
본 발명의 또 다른 측면은, 생 어성초 잎과 줄기 혼합 또는 생 어성초 뿌리를 상온 이하 온도의 저급 알코올에서 48시간 내지 120시간 냉침하여 추출하는 단계; 상기 알코올 추출물을 증류수에 1/100 내지 5/100 비율로 희석시키는 단계; 및 상기 희석된 알코올 추출물을 근육세포주 c2c12에 24시간 이상 전처리하는 단계;를 포함하는, 근감소 억제 방법을 제공한다.Another aspect of the present invention comprises the steps of extracting raw eoseongcho leaf and stem mixture or raw eoseongcho root in lower alcohol at a temperature below room temperature for 48 to 120 hours; diluting the alcohol extract in distilled water at a ratio of 1/100 to 5/100; and pre-treating the diluted alcohol extract with the muscle cell line c2c12 for at least 24 hours; it provides a method for inhibiting muscle loss, including.
상기 저급 알코올은 메탄올 또는 에탄올, 바람직하게는 식용 에탄올일 수 있다.The lower alcohol may be methanol or ethanol, preferably edible ethanol.
상기 알코올 추출은, 주정 추출을 포함할 수 있으며, 바람직하게는 상온 이하의 저온에서, 48시간 내지 120시간 추출할 경우, 근위축 유도된 근육세포의 세포 생존률을 가장 효과적으로 개선시킬 수 있다.The alcohol extraction may include alcohol extraction, and preferably at a low temperature below room temperature, when the extraction is performed for 48 to 120 hours, the cell viability of muscle atrophy-induced muscle cells can be most effectively improved.
일 측에 따를 때, 상기 알코올 추출 단계 이후, 감압농축 및 건조 분말화 단계;를 더 포함할 수 있다.According to one side, after the alcohol extraction step, concentration under reduced pressure and dry powdering step; may further include.
이하, 하기 실시예 및 비교예를 참조하여 본 발명을 상세하게 설명하기로 한다. 그러나, 본 발명의 기술적 사상이 그에 의해 제한되거나 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail with reference to the following Examples and Comparative Examples. However, the technical spirit of the present invention is not limited or limited thereto.
<제조예1. 아로니아 스톡 제조><Preparation Example 1. Aronia Stock Manufacturing>
아로니아(Aronia melanocarpa, 2020년산)는 전북 덕유산에서 재배, 수확된 것을 구입하였다. Aronia (Aronia melanocarpa, 2020) was grown and harvested in Deogyusan, Jeollabuk-do.
도 1을 참조할 때, 아로니아 258.188 g을 동결 건조한 39 g에 95% 에탄올 390 ml을 넣은 후 실온에 72시간 두었다가 원심 분리기에서 9,000 rpm으로 4℃, 15분간 원심 분리하였다.1, 258.188 g of Aronia 258.188 g was put in 39 g of freeze-dried 95% ethanol, 390 ml was placed at room temperature for 72 hours, and then centrifuged at 9,000 rpm in a centrifuge at 9,000 rpm for 15 minutes.
이후 상층액을 여과한 후 감압농축기 80℃에서 5분간 농축하여 액체형태의 추출물 6.5 mL을 얻어 시료로 사용하였다. 아로니아 추출물 스톡(stock)농도는 38.952 g/ml이다.Thereafter, the supernatant was filtered and concentrated for 5 minutes at 80° C. in a vacuum concentrator to obtain 6.5 mL of a liquid extract and used as a sample. The aronia extract stock concentration was 38.952 g/ml.
<실시예1><Example 1>
상기 제조예의 스톡을 증류수에 1/10,000 비율로 희석하여 3.895 mg/ml 농도로 희석하였다.The stock of Preparation Example was diluted in distilled water at a ratio of 1/10,000 to a concentration of 3.895 mg/ml.
<실시예2><Example 2>
상기 제조예의 스톡을 증류수에 1/1000 비율로 희석하여 38.950 mg/ml 농도로 희석하였다.The stock of Preparation Example was diluted to a concentration of 38.950 mg/ml by diluting it in distilled water at a ratio of 1/1000.
<실시예3><Example 3>
상기 제조예의 스톡을 증류수에 1/00 비율로 희석하여 389.50 mg/ml 농도로 희석하였다.The stock of Preparation Example was diluted with distilled water at a ratio of 1/00 to a concentration of 389.50 mg/ml.
<실시예4><Example 4>
상기 제조예의 스톡을 증류수에 1/1,000,000 비율로 희석하여 0.038 mg/ml 농도로 희석하였다.The stock of Preparation Example was diluted in distilled water at a ratio of 1/1,000,000 to a concentration of 0.038 mg/ml.
<실시예5><Example 5>
상기 제조예의 스톡을 증류수에 1/100,000 비율로 희석하여 0.389 mg/ml 농도로 희석하였다.The stock of Preparation Example was diluted to a concentration of 0.389 mg/ml by diluting it in distilled water at a ratio of 1/100,000.
<실험예1. 동물 실험><Experimental Example 1. Animal Testing>
실험 마우스의 좌골신경(sciatic nerve)를 절단한 후, 종아리 부위 근감소증을 유발시켰다. 비임상 실험으로 근감소증 유도 랫트에 직접 실시예 1의 아로니아 추출물을 투여하여 다양한 실험을 진행하였다.After cutting the sciatic nerve of the experimental mouse, calf sarcopenia was induced. As a non-clinical experiment, various experiments were conducted by administering the aronia extract of Example 1 directly to sarcopenia-induced rats.
진행한 실험은 근육의 힘 변화를 관찰하기 위한 grip strength과 Hanging time & Holding impulse 테스트, 근육손상 지표를 관찰하기 위한 혈액 분석, 근육을 분리하여 무게 측정 및 micro CT로 근육 크기 변화 관찰, 체중 및 섭식량 관찰, ELIZA TEST를 진행하였다.The experiments conducted were grip strength and hanging time & holding impulse tests to observe changes in muscle force, blood analysis to observe muscle damage indicators, weight measurement by separating muscles, and observation of changes in muscle size with micro CT, weight and food intake Observation and ELIZA TEST were performed.
1. 근육의 힘 변화 관찰1. Observation of changes in muscle strength
근육의 힘변화를 직접적으로 관찰하기 위하여 grip strength과 Hanging time & Holding impulse 테스트를 진행하였다.In order to directly observe the change in muscle force, grip strength, hanging time & holding impulse tests were performed.
하기의 도 5를 참조할 때, 대조군(sham)에 비해 신경절단 동물(NTX)에서 Grip strength가 감소하였고, 아로니아 추출물 투여 동물에서 Grip strength 가 증가함을 나타내어, 아로니아 추출물이 근육의 기능(power) 증가에 기여했음을 확인하였다.Referring to FIG. 5 below, compared to the control group (sham), the grip strength was decreased in the nerve amputated animals (NTX), and the grip strength increased in the animals administered with the aronia extract, indicating that the aronia extract was used for muscle function ( power) was confirmed to have contributed to the increase.
하기의 도 6을 참조할 때, 대조군(sham)에 비해 신경절단 동물에서 hanging time 및 holding impulse 가 감소하였고, 아로니아 추출물 투여 동물에서는 통계적으로 유의하지는 않았지만 두 지표 모두 증가하였다. 이를 통해, 아로니아 추출물이 근육의 기능 증가에 관여할 수 있음을 알 수 있다.Referring to FIG. 6 below, compared to the control group (sham), the hanging time and holding impulse were decreased in the ganglion amputated animals, and although not statistically significant in the animals administered with the aronia extract, both indicators increased. Through this, it can be seen that the aronia extract can be involved in the increase in muscle function.
2. 근육량 시각 관찰2. Visual observation of muscle mass
근육량을 시각적으로 관찰하기 위해, 종아리의 다양한 근육 분리하였다. 분리한 근육은, 전방경골근(TA: tibialis anterior), 장지지신근(EDLL: extensor digitorum longus), 대퇴사두근(QC: quadriceps), 비복근(GC: gastrocnemius) 이다. 분리한 각 근육의 무게를 측정하고, Lean / fat mass (n=3)측정하였으며, micro CT로 근육의 크기 변화를 관찰하였다. 그리고 식욕 증진제로의 효능을 관찰하기 위하여, 체중과 섭식량을 관찰하였다.To visually observe the muscle mass, various muscles of the calf were isolated. The separated muscles are anterior tibialis anterior (TA), extensor digitorum longus (EDLL), quadriceps (QC), and gastrocnemius (GC). The weight of each isolated muscle was measured, and lean / fat mass (n=3) was measured, and the change in muscle size was observed by micro CT. And in order to observe the efficacy as an appetite enhancer, body weight and intake were observed.
하기의 도 7을 참조할 때, 대조군(sham)에 비해 신경절단 동물에서 TA 근육무게가 감소하였고, 아로니아 투여 동물에서는 TA의 무게가 통계적으로 유의하게 증가함을 확인하였다. 이는 아로니아 추출물이 TA 근육의 크기(size) 증가에 관여했음을 의미하는 것이다.Referring to FIG. 7 below, compared to the control group (sham), the muscle weight of TA was decreased in the ganglioned animals, and it was confirmed that the weight of the TA was statistically significantly increased in the animals administered with aronia. This means that the aronia extract was involved in the increase in the size (size) of the TA muscle.
도 7의 TA 근육 사진을 참조할 때, 아로니아 추출물이 근육의 형태학적 크기(size) 증가에 관여함을 보여준다. 즉, TA 근육에서 효과가 크게 나타나는 걸로 보아 아로니아가 근육 형태별로 미치는 효과가 다를 수 있음을 의미한다.Referring to the TA muscle photograph of Figure 7, it shows that the aronia extract is involved in the increase in the morphological size (size) of the muscle. In other words, considering that the effect is large in the TA muscle, it means that the effect of aronia may be different for each muscle type.
하기의 도 8을 참조할 때, 신경절단 동물에서 감소한 lean mass가 아로니아 추출물 투여 동물에서는 증가하였고, 증가한 fat mass는 아로니아 추출물 투여 동물에서는 감소하였다. 유의할만한 큰 변화는 아니지만, 아로니아 추출물이 근육 및 지방 관련 기능 개선에 관여할 수 있음을 확인하였다.Referring to FIG. 8, the lean mass decreased in the ganglion amputated animals increased in the aronia extract-administered animals, and the increased fat mass decreased in the aronia extract-administered animals. Although not a significant change, it was confirmed that aronia extract could be involved in improving muscle and fat-related functions.
하기의 도 9를 참조할 때, 신경절단 동물 및 아로니아 추출물 투여 동물에서 체중 변화가 뚜렷하지 않았으나, 식욕은 대조군에 비해 증가하였음을 확인하였다. 이는, 아로니아 추출물이 식욕을 촉진하였을 가능성이 있고, 현재 근감소가 극심한 cachexia(악액질) 등의 치료에 식욕 촉진제가 사용되고 있는 상황에서 아로니아가 활용될 수 있음을 의미한다.Referring to FIG. 9 below, it was confirmed that the weight change was not evident in the ganglion amputated animals and the animals administered with the aronia extract, but the appetite increased compared to the control group. This means that aronia extract may have promoted appetite, and aronia can be utilized in a situation where an appetite stimulant is currently used for the treatment of cachexia (cachexia), where muscle loss is severe.
하기의 도 11을 참조할 때, micro CT 검사에서는 신경절단 동물에서 감소한 hindlimb 면적(붉은 선)이 아로니아 투여에 의해 유의한 수준으로 회복됨을 관찰하였다. 이는, 아로니아의 근감소 억제 효능이 micro CT 소견에서도 확인됨을 의미한다.Referring to FIG. 11 below, it was observed that the reduced hindlimb area (red line) in the ganglion amputated animals was restored to a significant level by the administration of aronia in the micro CT examination. This means that the muscle reduction inhibitory effect of aronia was confirmed in micro CT findings.
3. 혈액 테스트3. Blood test
근감소량 억제 효능에 관한 지표를 관찰하기 위해, 혈액 테스트를 진행하였고, 그 결과는 하기의 표 1과 같다.In order to observe the indicator regarding the muscle loss inhibitory effect, a blood test was performed, and the results are shown in Table 1 below.
Figure PCTKR2021019821-appb-img-000005
Figure PCTKR2021019821-appb-img-000005
표 1을 분석할 때, 신경절단 동물에서 증가한 LDH 및 CK가 아로니아 투여에 의해 유의한 수준으로 감소하였다. 또한 Glucose 와 BUN도 아로니아 추출물 투여 동물에서 감소함을 확인하였다. When analyzing Table 1, the increased LDH and CK in ganglioned animals were significantly reduced by Aaronia administration. In addition, it was confirmed that glucose and BUN were also decreased in animals treated with aronia extract.
상기 혈액 테스트를 통해, 아로니아가 근육 손상 지표인 LDH와 CK를 감소시킴을 확인하여 근감소 억제 효능에 대한 동물 혈액 수준에서 확인한 의의가 있다. 추가적으로 Glucose 와 BUN 억제 효능도 확인하였는데, 이는 아로니아가 혈당 감소 및 신장 보호 효능도 있음을 확인하였다.Through the blood test, it was confirmed that aronia reduces LDH and CK, which are indicators of muscle damage, and there is a significance confirmed in the animal blood level for the inhibitory effect on muscle loss. Additionally, Glucose and BUN inhibitory effects were also confirmed, which confirmed that aronia also has blood sugar lowering and renal protective effects.
4. ELISA TEST4. ELISA TEST
마이오스타틴 혈중 농도를 관찰하여 지표 분석을 하기위해 엘리자 테스트를 진행하였다.The ELISA test was performed to analyze the indicator by observing the myostatin blood concentration.
하기의 도 10을 참조할 때, 신경절단 동물에서 증가한 마이오스타틴 혈중 농도가 아로니아 투여에 의해 유의한 수준으로 감소함을 확인하였다. 이는, 아로니아의 근감소 억제 효능이 근육량 조절 표적 단백질인 마이오스타틴을 조절하였음을 의미한다.Referring to FIG. 10 below, it was confirmed that the increased myostatin blood concentration in ganglion amputated animals decreased to a significant level by Aaronia administration. This means that the muscle loss inhibitory effect of aronia regulated myostatin, a target protein for controlling muscle mass.
<실험예2. 아로니아 추출물을 이용한 세포실험><Experimental Example 2. Cell experiment using aronia extract>
아로니아 추출물을 골격근 세포주 C2C12에 처리 후 분석 및 관찰하였다.Aronia extract was analyzed and observed after treatment in the skeletal muscle cell line C2C12.
1. 아로니아 추출물의 세포 독성 확인1. Confirmation of Cytotoxicity of Aronia Extract
세포 생존율, 회복율을 분석하기 위하여 MTT 테스트를 진행하였다. The MTT test was performed to analyze the cell viability and recovery rate.
실시예 1 내지 5의 아로니아 추출물을 손상 유도하지 않은 세포주 C2C12에 처리하였으며, 그 결과는 도 12 그래프와 같다.The aronia extracts of Examples 1 to 5 were treated in the cell line C2C12 that did not induce damage, and the results are shown in the graph of FIG. 12 .
도 12에서 확인할 수 있는 바와 같이, 모든 농도의 아로니아 추출물은 C2C12 세포에 대해거의 세포 독성을 나타내지 아니하였다. 나아가 1/10,000 이상의 희석물을 처리하는 경우 오히려 생존한 세포의 수가 증가함을 확인할 수 있었다. 구체적으로 1/1,000 희석물을 처리하는 경우 16% 세포생존율이 증가하였으며, 1/100 희석물을 처리하는 경우 세포생존율이 80% 증가하였다.As can be seen in FIG. 12 , aronia extracts of all concentrations showed almost no cytotoxicity to C2C12 cells. Furthermore, it was confirmed that the number of surviving cells increased when the dilution of 1/10,000 or more was treated. Specifically, when the 1/1,000 dilution was treated, the cell viability increased by 16%, and when the 1/100 dilution was treated, the cell viability increased by 80%.
2. 아로니아 추출물의 근육 세포 보호 효과 확인2. Confirmation of muscle cell protective effect of aronia extract
C2C12 세포주에 실시예 1의 희석 추출물을 3시간 동안전처리한 후, 덱사메타손을 처리하여 손상을 유도하고 24시간 후 세포 생존율을 확인하였다. 그 결과는 도 2 그래프와 같다.C2C12 cell line was pre-treated with the diluted extract of Example 1 for 3 hours, dexamethasone was treated to induce damage, and cell viability was confirmed after 24 hours. The result is shown in the graph of FIG. 2 .
C2C12 세포주에 실시예 1의 희석 추출물을 24시간 동안전처리한 후, 덱사메타손을 처리하여 손상을 유도하고 24시간 후 세포 생존율을 확인하였다. 그 결과는 도 3 그래프와 같다.C2C12 cell line was pre-treated with the diluted extract of Example 1 for 24 hours, and then treated with dexamethasone to induce damage and check cell viability after 24 hours. The result is the same as the graph of FIG. 3 .
도 2 및 3을 참조할 때, 아로니아 추출물의 전처리는 근육 세포를 덱사메타손으로부터 보호함을 확인할 수 있었으며, 특히, 전처리 시간이 길수록 근손실 예방이 우수함을 확인할 수 있었다.Referring to Figures 2 and 3, it was confirmed that the pretreatment of the aronia extract protects the muscle cells from dexamethasone, and in particular, it can be confirmed that the longer the pretreatment time, the better the prevention of muscle loss.
3. 아로니아 추출물의 마이오스타틴 발현 억제 효과 확인3. Confirmation of inhibitory effect on myostatin expression of aronia extract
실시예 1, 실시예 4 및 5의 희석 추출물을 처리하여 근감소 억제 지표로 마이오스타틴의 발현 수준을 분석하기 위하여 Luciferase assay를 진행하였다. 구체적으로 Myostatin luciferase vector (pMSTN_Luc)가 과발현 되어있는 C2C12 세포주를 사용하였으며, MSTN_Luc-C2C12 세포를 seeding한 후 다음날 실시예 1, 실시예 4 및 5의 희석 추출물을 12시간 동안 처리하였다. 이어서, 1X PLB (1X passive lysis buffer)를 배양 배지에 첨가하여 세포를 용해시킨 후 luciferase assay전용 96well plate(white plate)에 세포용해물을 넣고, LAR Ⅱ시약을 넣은 후 플레이트를 기계(Multiplate enspire)에 넣어 활성을 측정하고 Bradford 시약을 이용하여 정량하였다.Luciferase assay was performed to analyze the expression level of myostatin as an indicator of muscle reduction inhibition by treating the diluted extracts of Examples 1, 4 and 5. Specifically, a C2C12 cell line in which Myostatin luciferase vector (pMSTN_Luc) is overexpressed was used, and after seeding MSTN_Luc-C2C12 cells, the diluted extracts of Examples 1, 4 and 5 were treated for 12 hours the next day. Then, 1X PLB (1X passive lysis buffer) was added to the culture medium to lyse the cells, and the cell lysate was put into a 96-well plate (white plate) for luciferase assay. The activity was measured and quantified using Bradford reagent.
하기의 도 13을 참조할 때, 아로니아 추출물을 농도별로 처리하면 1/10,000 희석 후 처리하면 62%까지 마이오스타틴 프로모터 활성 억제함을 확인하였다. 이는 아로니아의 근감소 억제가 마이오스타틴 프로모터 활성 억제에 의함을 의미한다.Referring to FIG. 13 below, it was confirmed that when the aronia extract was treated by concentration, the myostatin promoter activity was suppressed by 62% when treated after dilution of 1/10,000. This means that suppression of muscle loss of aronia is due to suppression of myostatin promoter activity.
4. 아로니아 추출물과 블루베리 추출물의 마이오스타틴 발현 억제 효능 비교4. Comparison of the inhibitory efficacy of myostatin expression of aronia extract and blueberry extract
상기 아로니아 추출물의 마이오스타틴 발현 억제 효과를 다른 베리류와 비교하고자 하였으며, 상술한 실험과 동일한 실험을 블루베리 추출물을 이용하여 수행하였다. 블루베리 추출물은 제조예 1의 아로니아 스톡 제조와 동일하게 블루베리 스톡을 제조하고 상기 스톡을 증류수에 1/100,000 비율로 희석하여 획득하였다. MSTN_Luc-C2C12 세포를 seeding한 후 다음날 실시예 5의 아로니아 추출물 또는 블루베리 추출물을 12 시간 동안 처리하고, 세포를 용해시킨 후 용해물을 96-well plate에 넣어 LARⅡ 시약을 넣고 루시퍼라아제의 활성을 측정하고 Bradford 시약을 이용하여 이를 정량하였다. 각 추출물의 처리량은 동일하다. To compare the myostatin expression inhibitory effect of the Aronia extract with other berries, the same experiment as the above-mentioned experiment was performed using a blueberry extract. The blueberry extract was obtained by preparing a blueberry stock in the same manner as in the preparation of the aronia stock of Preparation Example 1, and diluting the stock in distilled water at a ratio of 1/100,000. The next day after seeding MSTN_Luc-C2C12 cells, the aronia extract or blueberry extract of Example 5 was treated for 12 hours, the cells were lysed, the lysate was put into a 96-well plate, LARII reagent was added, and the activity of luciferase was measured and quantified using Bradford reagent. The throughput of each extract is the same.
그 결과, 블루베리 추출물도 골격근 세포에서 마이오스타틴의 발현을 억제하였으나, 아로니아 추출물이 블루베리보다 높은 수준으로 마이오스타틴의 발현을 억제함을 확인하였다(* p-value ≤ 0.05, ** p-value ≤ 0.01).As a result, it was confirmed that blueberry extract also suppressed myostatin expression in skeletal muscle cells, but aronia extract inhibited myostatin expression at a higher level than blueberry (* p-value ≤ 0.05, ** p-value ≤ 0.01).
<실험예3. 시아니딘 성분의 세포 실험><Experimental Example 3. Cell Experiment of Cyanidin Components>
아로니아 추출물의 주요 활성성분인 시아니딘을 골격근 세포주 C2C12에 처리 후 분석 및 관찰하였다.Cyanidin, the main active ingredient of the Aronia extract, was analyzed and observed after treatment in the skeletal muscle cell line C2C12.
1. 세포 생존율 분석1. Cell Viability Assay
세포 생존율, 회복율을 분석하기 위하여, MTT 테스트를 진행하였다. In order to analyze the cell viability and recovery rate, the MTT test was performed.
첫번째로 시아니딘 2 ug/ml, 0.1 mg/ml, 0.03 mg/ml, 0.01 mg/ml, 0.003 mg/ml를 준비하여 손상 유도한 세포주에 처리 후, 세포 생존율을 분석하였다.First, 2 ug/ml, 0.1 mg/ml, 0.03 mg/ml, 0.01 mg/ml, and 0.003 mg/ml of cyanidin were prepared and treated in the cell line to induce damage, and then the cell viability was analyzed.
그리고, 마이오스타틴 발현 정도를 관찰하기 위하여 시아니딘 2 ug/ml, 0.2 mg/ml, 0.02 mg/ml을 손상 유도한 세포주에 처리 후, 루시페라제 발현을 분석하였다.And, in order to observe the expression level of myostatin, 2 ug/ml, 0.2 mg/ml, 0.02 mg/ml of cyanidin was treated in the injury-induced cell line, and luciferase expression was analyzed.
하기의 도 14를 참조할 때, 아로니아 주요 성분인 시아니딘으로 골격근 세포주 C2C12의 생존율을 분석에서 2 ug/ml까지 세포독성 나타나지 않으며(왼쪽 그래프), 농도별로 6시간 처리 후 마이오스타틴 프로모터 활성이 0.02~2 ug/ml 범위에서 50% 감소함을 확인하였다(오른쪽 그래프).Referring to FIG. 14 below, in the analysis of the viability of the skeletal muscle cell line C2C12 with cyanidin, the main component of Aronia, cytotoxicity was not observed up to 2 ug/ml (left graph), and myostatin promoter activity after 6 hours of treatment at each concentration It was confirmed that 50% decrease in the 0.02~2 ug/ml range (right graph).
2. PCR 및 마이오스타틴 mRNA관찰2. PCR and myostatin mRNA observation
시아니딘을 0.2 ug/ml, 2 ug/ml 처리하여 PCR 돌린 후, 마이오스타틴 mRNA를 관찰하였다.Cyanidin was treated with 0.2 ug/ml, 2 ug/ml, and PCR was performed, and then, myostatin mRNA was observed.
하기의 도15를 참조할 때, 아로니아 주요 성분인 시아니딘 처리후 골격근 세포주 C2C12에서 myostatin mRNA의 발현이 농도의존적으로 억제됨을 확인하였다. 이는, 시아니딘이 마이오스타틴의 전사를 억제해서 근감소 억제함을 의미한다.15, it was confirmed that the expression of myostatin mRNA in the skeletal muscle cell line C2C12 was inhibited in a concentration-dependent manner after treatment with cyanidin, a major component of Aronia. This means that cyanidin inhibits the transcription of myostatin to suppress muscle loss.
3. 웨스턴 블롯3. Western Blot
시아니딘을 골격근 세포주 C2C12에 24시간 처리 후 웨스턴 블롯을 실시하였다.After 24 hours of treatment with cyanidin in the skeletal muscle cell line C2C12, Western blot was performed.
하기의 도 16을 참조할 때, 시아니딘 0.2 mg/ml를 투여했을 때, 세포성장 및 단백질 합성에 관여하는 mTOR 하위물질인 P70s6 kinase 와 akt/erk 인산화가 증가함을 확인하였다. 이는 아로니아의 근감소 억제 효과가 세포 내부 및 외부적 요인에 의한 근육세포 보호와 나아가 mTOR 신경전달 경로 활성화를 통한 근육섬유 형성에 관여하는 단백질의 합성을 유도하여 나타나는 것을 의미한다. 또한, 시아니딘은 Akt의 활성화를 통해 근육 분해를 촉진하는 Foxo1을 저해함으로써 근육 분해를 간접적으로 억제할 수 있음을 암시한다. Referring to FIG. 16 below, when 0.2 mg/ml of cyanidin was administered, it was confirmed that P70s6 kinase and akt/erk phosphorylation, which are sub-materials of mTOR involved in cell growth and protein synthesis, increased. This means that the muscle loss inhibitory effect of aronia appears by inducing the synthesis of proteins involved in muscle fiber formation through the protection of muscle cells by internal and external factors and further activation of the mTOR neurotransmission pathway. Furthermore, we suggest that cyanidin may indirectly inhibit muscle degradation by inhibiting Foxo1, which promotes muscle degradation through activation of Akt.
하기의 도 17을 참조할 때, 항암제 독소루비신 처리 후 근감소 유도된 모델에서, 시아니딘을 처리했을 때, 독소루비신에 의한 마이오스타틴 발현 증가가 억제됨을 확인하였다. 이는, 시아니딘이 항암제에 의해 발생하는 악액질(cachexia) 등을 억제하는 용도로 활용가능성이 있음을 암시한다.Referring to FIG. 17 below, it was confirmed that, in the muscle loss induced model after treatment with the anticancer drug doxorubicin, the increase in myostatin expression by doxorubicin was suppressed when cyanidin was treated. This suggests that cyanidin has potential for use in inhibiting cachexia, etc. caused by anticancer drugs.
하기의 도 18을 참조할 때, 덱사메타손으로 근감소 유도된 모델에서, 시아니딘을 처리하였을 때, 마이오스타틴 발현 증가가 억제됨을 확인하였다. 이는 시아니딘이 만성적인 스테로이드 치료에 의해 발생하는 쿠싱 증후군을 억제하는 용도로 활용가능성이 있음을 암시한다.Referring to FIG. 18 below, it was confirmed that, in the dexamethasone-induced muscle loss model, when cyanidin was treated, the increase in myostatin expression was suppressed. This suggests that cyanidin has potential for use in inhibiting Cushing's syndrome caused by chronic steroid treatment.
상기의 실시예1 내지 5와 비교예, 실험예 1 내지 3을 통한 실험에서 아로니아 추출물의 근육 무게 증가, 파워 증가 효과를 확인하였으며, 혈액에서 마이오스타틴 농도가 억제됨을 확인하였다. 또한 덱사메타손에 의한 세포 독성을 억제함을 확인하였으며, 마이오스타틴 프로모터 활성을 억제하고 마이오스타틴 mRNA발현이 억제됨을 확인하였다. 웨스턴 블롯을 통해 아로니아 추출물의 주요 활성성분이 세포성장/단백질 합성 관련 단백질 인산화를 증가시키며, 항암제/스테로이드에 의한 근감소를 억제함을 확인할 수 있었다.In the experiments through Examples 1 to 5, Comparative Examples, and Experimental Examples 1 to 3, the muscle weight increase and power increase effects of the aronia extract were confirmed, and it was confirmed that the myostatin concentration in the blood was suppressed. In addition, it was confirmed that the cytotoxicity caused by dexamethasone was suppressed, and it was confirmed that the myostatin promoter activity was suppressed and the myostatin mRNA expression was suppressed. Through Western blot, it was confirmed that the main active ingredient of the Aronia extract increased cell growth/protein synthesis-related protein phosphorylation and inhibited muscle loss caused by anticancer drugs/steroids.
즉, 아로니아의 근감소 억제 효능은 주성분인 시아니딘의 마이오스타틴 발현 억제, 세포성장 및 단백질 합성 기전 촉진에 의하며, 덱사메타손 및 독소루비신에 의한 마이오스타틴 발현증가를 억제함을 확인함으로써 스테로이드에 의한 쿠싱 증후군(cushing syndrom) 및 항암제 사용에 의한 암 악액질(cancer cachexia) 억제용으로 활용될 수 있다.That is, the muscle reduction inhibitory effect of Aronia is due to inhibition of myostatin expression of cyanidin, the main component, and promotion of cell growth and protein synthesis mechanism, and by confirming that it inhibits increase in myostatin expression caused by dexamethasone and doxorubicin. It can be utilized for suppressing Cushing's syndrome and cancer cachexia by using anticancer drugs.
본 발명은 아로니아 추출물을 유효성분으로 함유하는 근감소 억제 효능 조성물에 관한 것으로, 상기 아로니아 추출물은 분화를 유도한 근육세포 c2c12 세포주에 덱사메타손과 함께 투여시 근육세포 생존률이 90% 이상인 기능성 식품 조성물을 제공한다(실험예2의 세포 생존율 분석 실험). The present invention relates to a composition for inhibiting muscle loss containing an aronia extract as an active ingredient, wherein the aronia extract is a functional food composition having a muscle cell survival rate of 90% or more when administered together with dexamethasone to a muscle cell c2c12 cell line induced to differentiate. to provide (cell viability analysis experiment of Experimental Example 2).
동물실험 결과, 아로니아 추출물은 좌골 신경손상에 의한 근감소 유도 마우스의 근력회복은 물론 TA(tibialis anterior) 근육의 무게 감소 또한 억제하였음을 확인하였다(실험예1의 근육 힘 관찰 및 시각 관찰). As a result of the animal experiment, it was confirmed that the aronia extract inhibited not only the muscle strength recovery of mice induced by muscle loss due to sciatic nerve damage but also the weight loss of the tibialis anterior (TA) muscle (muscle strength observation and visual observation of Experimental Example 1).
또한, 신경 손상 모델에서 증가한 혈중 마이오스타틴 농도가 아로니아 투여군에서 감소함을 확인하여 아로니아의 근감소 억제효과가 마이오스타틴 조절에 의함을 밝혔다(실험예1의 ELIZA TEST). In addition, it was confirmed that the increased blood myostatin concentration in the nerve damage model decreased in the aronia-administered group, revealing that the muscle reduction inhibitory effect of aronia was due to myostatin control (ELIZA TEST of Experimental Example 1).
아로니아 주성분인 시아니딘이 근육양 조절 표적 단백질인 마이오스타틴의 발현양을 억제함을 확인하였다. 덱사메타존 및 항암제 독소루비신에 의한 마이오스타틴 단백질 발현 증가를 시아니딘이 억제함을 확인하였다(실험예3의 웨스턴 블롯). It was confirmed that cyanidin, the main component of Aronia, suppressed the expression level of myostatin, a target protein for controlling muscle mass. It was confirmed that cyanidin inhibited the increase in myostatin protein expression caused by dexamethasone and the anticancer drug doxorubicin (Western blot of Experimental Example 3).
본 발명에 따를 때, 상온의 에탄올에서 장시간 추출한 주정 추출 공정을 이용하여, 체내 반감기를 증대시키면서, 투여횟수 조절이 가능하고 약리학적 부작용이 적은 근감소 억제 효과를 기대할 수 있다. 또한 덱사메타존 장기간 사용에 의해 발생하는 쿠싱 증후군, 그리고 독소루비신 등 항암제에 의해 발생하는 악액질의 예방 및 치료에 활용될 수 있다. According to the present invention, by using the alcohol extraction process extracted in ethanol at room temperature for a long time, the half-life in the body can be increased, the frequency of administration can be controlled, and the effect of inhibiting muscle loss with few pharmacological side effects can be expected. In addition, it can be used for the prevention and treatment of Cushing's syndrome caused by long-term use of dexamethasone, and cachexia caused by anticancer drugs such as doxorubicin.
<제조예2. 어성초 추출물 제조><Preparation Example 2. Manufacture of Eoseongcho Extract>
어성초(Houttuynia Cordata Thunb)는 전북 군산에서 재배, 수확된 것을 구입하였다. Houttuynia Cordata Thunb was grown and harvested in Gunsan, Jeollabuk-do.
도 20을 참조할 때, 어성초는 잎과 줄기 혼합 25 g을 잘게 자른 후 80% 에탄올 750 mL에 96시간 냉침 후 여과지에 거른다. 원심 분리기에서 9,000 rpm으로 4℃, 15분간 원심 분리한 후 상층액을 여과한 후 감압농축기 80℃에서 5분간 농축하여 액체형태의 추출물 5 mL을 얻어 시료로 사용하였다.Referring to FIG. 20, after cutting 25 g of a mixture of leaves and stems, Eoseongcho is refrigerated in 750 mL of 80% ethanol for 96 hours and filtered through filter paper. After centrifugation at 9,000 rpm in a centrifuge for 15 minutes at 4°C, the supernatant was filtered and concentrated at 80°C in a vacuum concentrator for 5 minutes to obtain 5 mL of a liquid extract and used as a sample.
시료로 수득한 어성초 추출물을 증류수에 3/100 비율로 희석하여 기능성 식품 조성물의 유효성분으로 사용하였다.The extract obtained as a sample was diluted in distilled water at a ratio of 3/100 and used as an active ingredient in a functional food composition.
<실시예 1><Example 1>
근육세포주 C2C12에 상기 제조예에서 유효성분으로 수득한 희석한 어성초 추출물을 24시간 전처리하였다.The muscle cell line C2C12 was pre-treated with the diluted eoseongcho extract obtained as an active ingredient in the above Preparation Example for 24 hours.
이후, 근육세포주 C2C12에 희석한 어성초 추출물과 Dexamethasone 30uM을 동시에 다시 24시간 처리하여, 근감소증 유도하였다.Thereafter, eoseongcho extract diluted in the muscle cell line C2C12 and 30 uM of Dexamethasone were simultaneously treated again for 24 hours to induce sarcopenia.
<실시예 2><Example 2>
상기 제조예 및 실시예 1에서 어성초 잎과 줄기의 혼합 대신 어성초 뿌리를 사용한 것을 제외하고는 동일하게 실시되었다.It was carried out in the same manner as in Preparation Examples and Example 1, except that Eoseongcho root was used instead of a mixture of Eoseongcho leaves and stems.
<실시예 3><Example 3>
근육세포주 C2C12에 시판 어성초잎 건조 분말을 증류수와 3/100 농도로 희석하여 24시간 전처리한 후, 상기 시판 어성초잎 건조 분말을 3/100으로 희석한 추출물과 Dexamethasone 30uM을 동시에 24시간 처리하여, 근감소증 유도하였다.The muscle cell line C2C12 was diluted with distilled water to a concentration of 3/100 and pre-treated for 24 hours. nephropathy was induced.
<실험예 1. 근위축 유도된 근육세포의 세포 생존률 실험><Experimental Example 1. Cell viability test of muscle cells induced by muscle atrophy>
상기 실시예 1 내지 3에 상술한바와 같이 근육세포주 C2C12에 덱사메타손을 처리하여 근육 세포의 사멸을 유도하였으며, 어성초 추출물의 전처리에 따른 근육세포 생존율 변화를 MTT assay를 수행하여 확인하였다.As described above in Examples 1 to 3, the muscle cell line C2C12 was treated with dexamethasone to induce apoptosis of muscle cells, and the change in muscle cell viability according to the pretreatment of the Echo extract was confirmed by performing MTT assay.
하기의 도 21 및 23을 참조할 때, 실시예 1에 따른 어성초 잎과 줄기 혼합 추출물 희석액과 실시예 3에 따른 어성초 분말 희석액을 전처리하는 경우 근육세포를 보호하여 덱사메타손에 의한 근육세포 손상을 억제하였으며, 나아가 덱사메타손 처리에도 불구하고 오히려 근육양이 증가하는 것을 확인할 수 있었다. 도 21 내지 23 를 참조할 때, 어성초 추출물 희석액 및 어성초 분말 희석액 모두 근감소 억제 효과가 있고, 근위축 유도 없이 단독으로 처리시, 근육양이 증가하는 것을 확인할 수 있었다. 21 and 23 below, in the case of pre-treatment with the diluting eoseongcho leaf and stem mixed extract according to Example 1 and the dilute eosinwort powder according to Example 3, the muscle cells were protected and muscle cell damage caused by dexamethasone was inhibited. , and furthermore, it was confirmed that the muscle mass rather increased despite the dexamethasone treatment. Referring to FIGS. 21 to 23 , both the dilute eoseongcho extract and the diluted eoseongcho powder have a muscle reduction inhibitory effect, and when treated alone without inducing muscle atrophy, it was confirmed that the amount of muscle increased.
<실험예 2. 어성초 추출물의 마이오스타틴 발현 억제 효과 확인><Experimental Example 2. Confirmation of the myostatin expression inhibitory effect of Eoseongcho extract>
이어서, 어성초 추출물이 근감소 억제 지표인 마이오스타틴의 발현에 미치는 영향을 분석하기 위하여 Luciferase assay를 진행하였다. 구체적으로, 제조예의 어성초 추출물과 실시예 3의 어성초 잎 건조 분말 추출물을 증류수와 혼합하여 1/100으로 희석하여 각 어성초 추출물을 준비하였다. 이어서, Myostatin luciferase vector (pMSTN_Luc)가 과발현 되어있는 C2C12 세포주를 seeding하고 다음날 먼저 준비한 각 어성초 추출물을 처리하였다. 12시간 후에 1X PLB (1X passive lysis buffer)를 배양 배지에 첨가하여 세포를 용해시킨 후 luciferase assay전용 96well plate(white plate)에 세포용해물을 넣고, LAR Ⅱ시약을 넣은 후 플레이트를 기계(Multiplate enspire)에 넣어 활성을 측정하고 Bradford 시약을 이용하여 정량하였다.Next, Luciferase assay was performed to analyze the effect of the eosinth extract on the expression of myostatin, an indicator of muscle loss inhibition. Specifically, each eoseongcho extract was prepared by mixing the eoseongcho extract of Preparation Example and the dried eoseongcho leaf dry powder extract of Example 3 with distilled water and diluting 1/100. Next, the C2C12 cell line overexpressing the Myostatin luciferase vector (pMSTN_Luc) was seeded and treated with each Ewoongcho extract prepared in advance the next day. After 12 hours, 1X PLB (1X passive lysis buffer) was added to the culture medium to lyse the cells, and then the cell lysate was put in a 96-well plate (white plate) dedicated to luciferase assay, and LAR II reagent was added, and the plate was then machined (Multiplate enspire). ) to measure the activity and quantified using Bradford reagent.
그 결과, 도 24에서 확인할 수 있는 바와 같이 어성초 추출물의 처리시 마이오스타틴 프로모터 활성 억제함을 확인하였다. 이는 어성초 추출물의 근감소 억제가 마이오스타틴 프로모터 활성 억제에 의함을 의미한다.As a result, as can be seen in FIG. 24 , it was confirmed that the myostatin promoter activity was suppressed during the treatment of the Ewoseongcho extract. This means that the suppression of muscle loss of the Eosongcho extract is due to suppression of myostatin promoter activity.
이상과 같이 실시예들이 비록 한정된 도면에 의해 설명되었으나, 해당 기술분야에서 통상의 지식을 가진 자라면 상기를 기초로 다양한 기술적 수정 및 변형을 적용할 수 있다. 예를 들어, 설명된 기술들이 설명된 방법과 다른 순서로 수행되거나, 및/또는 설명된 시스템, 구조, 장치, 회로 등의 구성요소들이 설명된 방법과 다른 형태로 결합 또는 조합되거나, 다른 구성요소 또는 균등물에 의하여 대치되거나 치환되더라도 적절한 결과가 달성될 수 있다.As described above, although the embodiments have been described with reference to the limited drawings, those skilled in the art may apply various technical modifications and variations based on the above. For example, the described techniques are performed in an order different from the described method, and/or the described components of the system, structure, apparatus, circuit, etc. are combined or combined in a different form than the described method, or other components Or substituted or substituted by equivalents may achieve an appropriate result.
그러므로, 다른 구현들, 다른 실시예들 및 특허청구범위와 균등한 것들도 후술하는 청구범위의 범위에 속한다.Therefore, other implementations, other embodiments, and equivalents to the claims are also within the scope of the following claims.
본 발명은 근육세포 보호, 근 기능 개선, 및 근량 증가 효과를 갖는 조성물에 관한 것으로서 천연물 유래의 성분을 이용하여 근육질환 예방 및 치료를 위한 의약품, 의약외품에 이용될 수 있으며, 근 기능 개선 및 근량 증가를 위한 식품, 건강기능식품, 향장소재 등에 이용될 수 있다.The present invention relates to a composition having the effect of protecting muscle cells, improving muscle function, and increasing muscle mass, and can be used in pharmaceuticals and quasi-drugs for the prevention and treatment of muscle diseases using ingredients derived from natural products, improving muscle function and increasing muscle mass It can be used as food, health functional food, cosmetic material, etc.

Claims (25)

  1. 아로니아 추출물 또는 어성초 추출물을 유효성분으로 함유하는, 근육 질환 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating muscle disease, containing an aronia extract or eoseongcho extract as an active ingredient.
  2. 제1항에 있어서, According to claim 1,
    상기 아로니아 추출물은 하기 화학식 1의 시아니딘 배당체 또는 이의 약학적으로 유효한 염을 유효성분으로 함유하는, 약학적 조성물:The aronia extract contains a cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient, a pharmaceutical composition:
    Figure PCTKR2021019821-appb-img-000006
    .
    Figure PCTKR2021019821-appb-img-000006
    .
  3. 제1항에 있어서, The method of claim 1,
    상기 아로니아 추출물의 농도는 0.1 mg/ml 내지 1000 mg/ml 인, 약학적 조성물.The concentration of the aronia extract is 0.1 mg / ml to 1000 mg / ml, the pharmaceutical composition.
  4. 제1항에 있어서, According to claim 1,
    상기 아로니아 추출물은 젖산탈수소효소(LDH), 크레아틴키나제(CK) 또는 이 둘 모두의 혈중 농도를 감소시키는, 약학적 조성물.The aronia extract is lactate dehydrogenase (LDH), creatine kinase (CK), or reducing the blood concentration of both, a pharmaceutical composition.
  5. 제1항에 있어서, The method of claim 1,
    상기 조성물은 근육 단백질을 파괴하는 마이오스타틴(Myostatin)의 발현을 억제하는, 약학적 조성물.The composition inhibits the expression of myostatin (Myostatin) that destroys muscle protein, a pharmaceutical composition.
  6. 제1항에 있어서, According to claim 1,
    상기 아로니아 추출물은 mTOR를 포함하는 세포 내 단백질 합성 기전을 활성화시키는, 약학적 조성물.The aronia extract is a pharmaceutical composition that activates an intracellular protein synthesis mechanism including mTOR.
  7. 제1항에 있어서, According to claim 1,
    상기 어성초 추출물은 어성초의 잎, 줄기, 및 뿌리로 이루어진 군으로부터 선택된 1종 이상의 부위로부터 추출된 것인, 약학적 조성물.The eoseongcho extract will be extracted from one or more parts selected from the group consisting of leaves, stems, and roots of eoseongcho, a pharmaceutical composition.
  8. 제1항에 있어서, According to claim 1,
    상기 아로니아 추출물 또는 어성초 추출물은 물, C1내지 C4의 저급알코올 및 이들의 혼합용매로 이루어진 군으로부터 선택된 하나 이상의 용매로 추출된 것인, 약학적 조성물.The aronia extract or Eoseongcho extract is water, C 1 to C 4 of the lower alcohol, and a pharmaceutical composition that is extracted with one or more solvents selected from the group consisting of mixed solvents thereof.
  9. 제1항에 있어서, According to claim 1,
    상기 어성초 추출물은 냉침 추출 방법으로 48시간 내지 120시간 동안 추출된 것인, 약학적 조성물.The eoseongcho extract is extracted for 48 hours to 120 hours by the cold-chim extraction method, a pharmaceutical composition.
  10. 제1항에 있어서,According to claim 1,
    상기 근육 질환은 근 기능 저하, 근육 감소, 근육 위축, 근육 소모 또는 근육 퇴화로 인한 근육 질환인 것인, 약학적 조성물.The muscle disease is a muscle disease due to decreased muscle function, muscle loss, muscle atrophy, muscle wasting or muscle degeneration, a pharmaceutical composition.
  11. 제1항에 있어서,According to claim 1,
    상기 근육 질환은 긴장감퇴증(atony), 근위축증(muscular atrophy), 근이영양증(muscular dystrophy), 근무력증, 악액질(cachexia), 경직성 척추 증후군(rigid spinesyndrome), 근위축성 측삭경화증(루게릭병, amyotrophic lateral sclerosis), 경직성 척추 증후군(rigid spinsesyndrome), 샤르코-마리-투스병 (Charcot-Marie-Tooth disease) 및 근육 감소증(sarcopenia)으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것인, 약학적 조성물.The muscle disease is dystonia, muscular atrophy, muscular dystrophy, myasthenia gravis, cachexia, rigid spinesyndrome, amyotrophic lateral sclerosis (Lou Gehrig's disease, amyotrophic lateral sclerosis). , Rigid spinsesyndrome, Charcot-Marie-Tooth disease (Charcot-Marie-Tooth disease), and any one or more selected from the group consisting of sarcopenia (sarcopenia), the pharmaceutical composition.
  12. 하기 화학식 1의 시아니딘 배당체 또는 이의 약학적으로 유효한 염을 유효성분으로 함유하는, 근육 질환 예방 또는 치료용 약학적 조성물:A pharmaceutical composition for preventing or treating muscle disease, comprising as an active ingredient a cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof:
    Figure PCTKR2021019821-appb-img-000007
    .
    Figure PCTKR2021019821-appb-img-000007
    .
  13. 제12항에 있어서, 13. The method of claim 12,
    상기 시아니딘 배당체는 또는 이의 염은 아로니아 유래의 것인, 약학적 조성물.The cyanidin glycoside or a salt thereof is derived from Aronia, a pharmaceutical composition.
  14. 제12항에 있어서, 13. The method of claim 12,
    상기 시아니딘 배당체 또는 이의 염은 근육 단백질을 파괴하는 마이오스타틴(Myostatin)의 발현을 억제하는, 약학적 조성물.The cyanidin glycoside or salt thereof inhibits the expression of myostatin, which destroys muscle protein, a pharmaceutical composition.
  15. 제12항에 있어서,13. The method of claim 12,
    상기 시아니딘 배당체 또는 이의 염은 mTOR를 포함하는 세포 내 단백질 합성 기전을 활성화시키는, 약학적 조성물.The cyanidin glycoside or a salt thereof activates an intracellular protein synthesis mechanism including mTOR, a pharmaceutical composition.
  16. 제12항에 있어서, 13. The method of claim 12,
    상기 근육 질환은 근 기능 저하, 근육 감소, 근육 위축, 근육 소모 또는 근육 퇴화로 인한 근육 질환인 것인, 약학적 조성물.The muscle disease is a muscle disease due to decreased muscle function, muscle reduction, muscle atrophy, muscle wasting or muscle degeneration, a pharmaceutical composition.
  17. 제12항에 있어서,13. The method of claim 12,
    상기 근육 질환은 긴장감퇴증(atony), 근위축증(muscular atrophy), 근이영양증(muscular dystrophy), 근무력증, 악액질(cachexia), 경직성 척추 증후군(rigid spinesyndrome), 근위축성 측삭경화증(루게릭병, amyotrophic lateral sclerosis), 경직성 척추 증후군(rigid spinsesyndrome), 샤르코-마리-투스병 (Charcot-Marie-Tooth disease) 및 근육 감소증(sarcopenia)으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것인, 약학적 조성물.The muscle disease is dystonia, muscular atrophy, muscular dystrophy, myasthenia gravis, cachexia, rigid spinesyndrome, amyotrophic lateral sclerosis (Lou Gehrig's disease, amyotrophic lateral sclerosis). , Rigid spinsesyndrome, Charcot-Marie-Tooth disease (Charcot-Marie-Tooth disease), and any one or more selected from the group consisting of sarcopenia (sarcopenia), the pharmaceutical composition.
  18. 아로니아 추출물 또는 어성초 추출물을 유효성분으로 함유하는, 근 기능 개선용 식품 조성물.A food composition for improving muscle function, which contains an aronia extract or an eosinosa extract as an active ingredient.
  19. 하기 화학식 1의 시아니딘 배당체 또는 이의 약학적으로 유효한 염을 유효성분으로 함유하는, 근 기능 개선용 식품 조성물:A food composition for improving muscle function, containing a cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient:
    Figure PCTKR2021019821-appb-img-000008
    Figure PCTKR2021019821-appb-img-000008
  20. 아로니아 추출물 또는 어성초 추출물을 유효성분으로 함유하는, 근육 양(muscle mass) 증가 또는 근육 감소 억제 식품 조성물.A food composition containing an aronia extract or an eosinosa extract as an active ingredient, increasing muscle mass or inhibiting muscle loss.
  21. 하기 화학식 1의 시아니딘 배당체 또는 이의 약학적으로 유효한 염을 유효성분으로 함유하는, 근육 양(muscle mass) 증가 또는 근육 감소 억제용 식품 조성물:A food composition for inhibiting muscle mass increase or muscle loss, containing a cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof as an active ingredient:
    Figure PCTKR2021019821-appb-img-000009
    .
    Figure PCTKR2021019821-appb-img-000009
    .
  22. 아로니아 추출물 또는 어성초 추출물을 개체에 투여하는 단계를 포함하는, 근육 질환 예방 또는 치료 방법.A method for preventing or treating muscle disease, comprising administering an aronia extract or an extract of eosinopsis to an individual.
  23. 근육 질환 예방 또는 치료용 약제 제조를 위한 아로니아 추출물 또는 어성초 추출물의 용도.The use of an aronia extract or a kelp extract for the manufacture of a medicament for the prevention or treatment of muscle disease.
  24. 하기 화학식 1의 시아니딘 배당체 또는 이의 약학적으로 유효한 염을 개체에 투여하는 단계를 포함하는, 근육 질환 예방 또는 치료 방법:A method for preventing or treating muscle disease, comprising administering to an individual a cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof:
    Figure PCTKR2021019821-appb-img-000010
    .
    Figure PCTKR2021019821-appb-img-000010
    .
  25. 근육 질환 예방 또는 치료용 약제 제조를 위한 하기 화학식 1의 시아니딘 배당체 또는 이의 약학적으로 유효한 염의 용도:Use of a cyanidin glycoside of Formula 1 or a pharmaceutically effective salt thereof for the preparation of a medicament for the prevention or treatment of muscle disease:
    Figure PCTKR2021019821-appb-img-000011
    .
    Figure PCTKR2021019821-appb-img-000011
    .
PCT/KR2021/019821 2020-12-24 2021-12-24 Composition for preventing or treating muscle diseases containing aronia or houttuynia cordata extract as active ingredient WO2022139544A1 (en)

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KR1020210108529A KR20220092346A (en) 2020-12-24 2021-08-18 A composition comprising extracts of aronia for inhibiting muscle atrophy
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KR1020210183652A KR20220092401A (en) 2020-12-24 2021-12-21 A composition comprising extracts of aronia for muscle diseases
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011108487A1 (en) * 2010-03-01 2011-09-09 国立大学法人九州大学 Muscular atrophy inhibitor
KR101372037B1 (en) * 2010-04-06 2014-03-10 (주)아모레퍼시픽 Composition for Promotion of transformation of muscle type

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011108487A1 (en) * 2010-03-01 2011-09-09 国立大学法人九州大学 Muscular atrophy inhibitor
KR101372037B1 (en) * 2010-04-06 2014-03-10 (주)아모레퍼시픽 Composition for Promotion of transformation of muscle type

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
OSZMLANSKL JAN, SAPIS JEAN C: "Anthocyanins in Fruits of Aronia Melanocarpa (Chokeberry)", REPRINTED FROM J. FOOD SCI, vol. 53, no. 4, 1 January 1988 (1988-01-01), pages 1241 - 1242, XP055945401 *
PAN-JUN SHIN, YEON-HUI KIM, IN-SOO LIM: "The effect of aronia, montmorency, and prunus mume upplementation on blood fatigue factors and muscle damage enzymes after running 10 km", JOURNAL OF EXERCISE NUTRITION & BIOCHEMISTRY, vol. 16, no. 1, 1 March 2012 (2012-03-01), pages 19 - 26, XP009537900, ISSN: 2233-6834, DOI: 10.5717/jenb.2011.16.1.19 *
PARK, HYE-MI;HONG, JOO-HEON;: "Physiological activities of Aronia melanocarpa extracts on extraction solvents", vol. 21, no. 5, 30 October 2014 (2014-10-30), pages 718 - 726, XP053034160 *

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