KR102677572B1 - A composition comprising an extract of Alpinia officinarum for treating and preventing skeleton muscle-related disorder - Google Patents

Abstract

The present invention relates to a composition for preventing and treating skeletal muscle-related diseases containing galangal extract or compounds isolated therefrom as active ingredients. Through (1) an effect experiment (Experimental Examples 1 and 5) on myoblast differentiation using the galangal extract and compounds of the present invention, cylinder-shaped multinucleated myotube cells (multinucleated) were obtained by treatment of the samples. It was confirmed that differentiation into myotubes was induced in a concentration-dependent manner, and the number of multinucleated myotubes increased. (2) Mechanism study of the p38 MAPK signaling system for promoting myoblast differentiation (Experimental Example 2); (3) Through experiments on the inhibition of muscle loss with the galangal extract and the compounds isolated therefrom in an in vitro model of muscle loss (Experimental Examples 3, 4, and 6), it was found that the samples of the present invention promoted myogenic cell differentiation and myotubes. Confirmed to have cell protective effects, it has been confirmed to treat atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic axonal sclerosis, myasthenia gravis, cachexia, and sarcopenia. By confirming that it can be used as a treatment or supplement for skeletal muscle diseases, it was confirmed that the composition is useful as a pharmaceutical composition for preventing and treating skeletal muscle diseases, health functional food, and health supplements.

Description

A composition comprising an extract of Alpinia officinarum for treating and preventing skeletal muscle-related disorder}

The present invention relates to a composition for preventing and treating skeletal muscle-related diseases containing galangal extract or a compound isolated therefrom.

[Document 1] J Cachexia Sarcopenia Muscle. 2015, 6, 197

[Document 2] Pharmacol Res. 2015, 99 , 86)

[Document 3] Pharmacol. Res. 2015, 99, 86-100

[Document 4] The Korea Journal of Sports Science 2011, 30, 1551

[Document 5] Cell Mol Life Sci 70: 4117-4130, 2013

[Document 6] J. Biol. Chem. 2002, 277, 49831

[Document 7] Korean J Pharmacogn. 2006, 37 , 56-59

[Document 8] Chem. Pharm. Bull. 1982, 30 , 2279-2282

[Document 9] J Med. Food. 2009, 13 , 1235

[Document 10] J Med. Food. 2012, 15 , 2286

[Document 11] J Nat. Prod. 2002, 65 , 1315-1318

[Document 12] Planta Med . 2006, 72 , 68-71

[Document 13] Fitoterapia 2008, 79 , 27-31

[Document 14] J Ethnopharmacol . 2018, 224, 45-62.

[Document 15] Anticancer Res. 2009, 29, 4981-4988;

[Document 16] Chem. Pharm. Bull. 1981, 29: 2383-2385

[Document 17] Chem. Pharm. Bull . 1985, 33, 4889-4893

[Document 18] Arch. Pharmacal Res. 2100, 34: 1289-1296

[Document 19] Chem. Pharm. Bull. 1982, 30: 2279-2282

[Document 20] Planta Med. 2008, 74: 427-431

[Document 21] Magn. Reson. Chem. 2001, 39, 374-380

[Document 22] Chem. Biol. Interact. 2016, 248, 60

[Document 23] Trends Cell Biol . 2006, 16 , 36

[Document 24] Mol. Biol. Cell. 2010, 21, 2399

[Document 25] Int. J. Mol. Med. 2015, 36 , 29-42

[Document 26] Biomed. Pharm. 2017, 95 , 1486

The present invention relates to a composition for preventing and treating skeletal muscle-related diseases containing galangal extract or a compound isolated therefrom.

Muscles can be divided into the following three types in terms of structure and function: (1) skeletal muscles located directly under the skin in the hands, feet, chest, stomach, and back and attached between bones, (2) heart. These include the myocardium, which forms the wall, and (3) the internal muscles, which form the walls of the stomach, bladder, uterus, etc. ([Naver Knowledge Encyclopedia] Types of Muscles (Doosan Encyclopedia))

Muscle regeneration can be used to treat atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic axonal sclerosis, myasthenia gravis, cachexia, and sarcopenia. It has been attracting attention for the purpose of overcoming skeletal muscle degenerative diseases such as. ( J Cachexia Sarcopenia Muscle. 2015, 6, 197)

Progressive muscle weakness and loss of function threatens quality of life and reduces the survival rate of cancer patients. More than 30% of cancer patients die due to weight loss due to muscle loss. This loss of muscle function is caused by degeneration of myo-proteins and decreases in muscle fiber cross-sectional area, muscle strength, nuclear number of myofibers, and insulin responsiveness. It is commonly accompanied by etc.

In addition to cancer, muscle loss can also be caused by the aging process and various chronic diseases. As aging progresses, part of the newly created skeletal muscle is replaced by fibrous tissue, resulting in sarcopenia, a decrease in the body's skeletal muscle mass and strength. Additionally, muscle loss also occurs in chronic diseases whose incidence increases with age, such as high blood pressure, impaired glucose tolerance, diabetes, obesity, dyslipidemia, atherosclerosis, and cardiovascular disease. ( Pharmacol Res. 2015 , 99 , 86).

Muscle dystrophy treatment strategies include inhibition of inflammatory molecules and myostatin or activation of cyclic AMP, proliferator-activated receptor gamma coactivator (PGC)-1α, and insulin signaling system. Although many potential drugs have been developed, megestrol acetate is the only treatment for muscular dystrophy approved by the U.S. Food and Drug Administration. Drugs for the treatment and prevention of muscle dystrophy have the activity of inhibiting muscle protein catabolism or promoting satellite cell functions. ( Pharmacol. Res. 2015 , 99, 86-100).

In muscles, anabolism and catabolism are balanced to regulate muscle production, and at this time, various biological signaling processes are regulated in relation to this within muscle cells. When a signaling reaction that induces the synthesis rather than the breakdown of muscle proteins is activated, the synthesis of muscle proteins increases, leading to an increase in muscle size (hypertrophy) or an increase in the number of muscle fibers (hyperplasia), resulting in excessive muscle production (The Korea Journal of Sports Science 2011, 30, 1551).

Myoblast cell differentiation and muscle formation are regulated by various factors (cell Mol Life Sci 70: 4117-4130, 2013). Among them, myoD initiates the expression of specific genes related to muscle differentiation and induces mesenchymal stem cells to differentiate into myoblasts. Myogenin and MHC (myosin heavy chain) regulated by MyoD induce fusion of myogenic cells, leading to the formation of myotubes and muscle fibers. Muscle fibers formed through this process form bundles and ultimately form muscles (Cell Mol Life Sci 70: 4117-4130, 2013)

Under normal conditions, quiescent satellite cells, known as primary stem cells, continuously proliferate and differentiate. Damaged muscles secrete various growth factors that activate the proliferation of myogenic satellite cells, referred to as myoblasts. Activated myogenic cells induce myogenic factors such as Myo D, Myf (myogenic factor)-5, myogenin, and Mrf-4. MyoD and Myf-5 are transcription factors specifically expressed in the myogenic lineage and play an important role in the initiation of myogenic cell differentiation. (Langley, B.; Thomas, M.; Bishop, A.; Sharma, M.; Gilmour, S.; Kambadur, R. J. Biol. Chem. 2002, 277, 49831). In particular, Myo D induces the expression of myogenic proteins such as MHC and myogenin through binding to non-muscle specific factors such as E protein, Mef-2 family proteins, and transcription cofactors.

Alpinia officinarum is a rhizome of a perennial herbaceous plant belonging to the Zingiberaceae family, and is also called Goryanggang. The roots and stems are used as medicinal ingredients, and contain essential oil components such as 1,8-cinerol, methyl-cinnamate, eugenol, pinene, and α-cadinene, as well as flavonoids such as galangin, kampferide, and many diarylheptanoid compounds. (Professor Bonchohak 2000). In East Asia, it has traditionally been used for gastrointestinal diseases such as cold stomach pain, vomiting, and abdominal pain, as well as rheumatic diseases (Jong-pil Lim, 2003). Pharmacological effects of Yang Gang include blood pressure lowering (Kim, Yoo et al. 2006), prostaglandin inhibition (Kiuchi, Shibuya et al. 1982), and anti-obesity (Xia, Yu et al. 2010; Jung, Jang et al. 2012), anti-emetic action (Shin, Kinoshita et al. 2002), anti-inflammatory action (Lee, Kim et al. 2006), and anti-cancer action (An, Zou et al. 2008). The German Commission E Monographs list it as being used for loss of appetite and indigestion, and the US FDA defines it as ‘generally regarded as safe (21CFR sections 182. 10, 182. 20).’

In particular, it has been reported that diarylheptanoids isolated from Yanggang exhibit anti-inflammatory (Abubakar, I. B., et al. 2018) activity and anti-cancer activity by inhibiting the cancer cell cycle and inducing apoptosis (Tabata, Yamazaki et al. 2009).

However, nowhere in the above literature is there any disclosure or teaching about the therapeutic effect of skeletal muscle-related diseases containing galangal extract as an active ingredient.

Accordingly, in order to develop an effective preventive and therapeutic agent for skeletal muscle-related diseases, the present inventors conducted (1) experiments on the effect of extracts and compounds on myoblast differentiation (Experimental Examples 1 and 5) to treat samples. Differentiation into cylinder-shaped multinucleated myotubes was induced in a concentration-dependent manner, and the number of multinucleated myotubes was confirmed to increase. (2) Promoting myoblast differentiation p38 MAPK signaling system mechanism research experiment (Experimental Example 2); (3) Through experiments on the inhibition of muscle loss with the galangal extract and compounds isolated therefrom in an in vitro model of muscle loss (Experimental Examples 3, 4, and 6), it was found that the samples of the present invention differentiated into muscle cells. It was confirmed that it not only promotes but also inhibits muscle loss in a muscle injury model. The present invention was completed after confirming that galangal extract and galangal-derived compounds can be used as a treatment or adjuvant for skeletal muscle diseases.

The object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of skeletal muscle-related diseases, containing galangal extract or a compound isolated therefrom as an active ingredient.

In addition, the present invention provides a health functional food for preventing and improving skeletal muscle-related diseases containing galangal extract or a compound isolated therefrom as an active ingredient.

Yanggang (referred to as Goryanggang or Xinjiang), as defined herein, is the root of congeners such as Alpinia officinarum, Alpinia galanga, Galanga, and Galangal. , roots, stems, sentinel leaves, preferably rhizomes, roots or sentinels.

The Yangjiang extract as defined herein is characterized as comprising a Yangjiang crude extract, a polar solvent-soluble extract purified from the Yangjiang crude extract, or a non-polar solvent-soluble extract.

The crude extract defined herein is a solvent selected from water including purified water, lower alcohols with 1 to 4 carbon atoms such as methanol, ethanol, butanol, or mixed solvents thereof, preferably water or a mixed solvent of water and ethanol, more preferably It is characterized as an extract soluble in ethanol.

Non-polar solvent-soluble extract fractions, as defined herein, include non-polar solvent-soluble extract fractions obtained by purifying only the extracts soluble in hexane, methylene chloride, chloroform, or ethyl acetate solvents from the crude extract herein.

The polar solvent-soluble extract as defined herein includes the extraction fraction soluble in a solvent selected from water, methanol, butanol, or a mixed solvent thereof remaining after removing the non-polar solvent-soluble fractions from the crude extract.

The compound isolated from galangal extract as defined herein is 7-(4-hydroxy-3-methoxyphenyl)-1-phenylhept-4-en-3-one [7-(4-hydroxy-3-methoxyphenyl) -1-phenylhept-4-en-3-one, compound 1], 5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenylheptan-3-one [5-hydroxy- 7-(4-hydroxy-3-methoxyphenyl)-1-phenylheptan-3-one, compound 2], 5-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)heptan-3- one [5-Hydroxy-1,7-bis(4-hydroxy-3- methoxyphenyl)heptan-3-one, compound 3], 1,7-diphenylhept-4-en-3-one [1,7- diphenylhept-4-en-3-one, compound 4], 1,7-diphenyl-5-hydroxy-3-heptanone [1,7-diphenyl-5-hydroxy-3-heptanone, compound 5], 5 -Hydroxy-7-(4''-hydroxyphenyl)-1-phenyl-3-heptanone [5-hydroxy-7-(4''-hydroxyphenyl)-1-phenyl-3-heptanone, compound 6] , 1,7-diphenyl-5-methoxy-3-heptanone [1,7-diphenyl-5-methoxy-3-heptanone, Compound 7], 3,5,7-trihydroxy-2-phenyl- Includes chromen-4-one [3,5,7-Trihydroxy-2-phenyl-chromen-4-one, compound 8].

High performance liquid chromatography (HPLC) analysis of Yangjiang extracts and compounds as defined herein is performed under conditions in which a non-polar solvent such as methanol or acetenitrile is mixed with an acidic aqueous solvent.

Skeletal muscle-related diseases defined herein include atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic axonal sclerosis, myasthenia gravis, cachexia, and senile sarcopenia ( at least one muscle disease selected from the group consisting of sarcopenia, specifically, skeletal muscle muscle-related disease caused by age-related muscular atrophy or cancer, more specifically, muscular atrophy due to age-related muscular atrophy or cancer, and muscular dystrophy ), muscle degeneration, muscle stiffness, amyotrophic axonal sclerosis, myasthenia gravis, cachexia, sarcopenia, and muscle loss.

In addition, the present invention provides a preventive and therapeutic agent for skeletal muscle-related diseases caused by cancer or an adjuvant anticancer therapeutic agent containing a galangal extract or a compound isolated therefrom.

In addition, the present invention provides a pharmaceutical composition, anticancer treatment supplement, or health functional food for the prevention and treatment of skeletal muscle-related diseases caused by cancer, which contains a combination of galangal extract or a compound isolated therefrom and a conventional anticancer agent as an active ingredient.

Existing anticancer drugs defined herein include Cyclophosphamide, methotrexate, 5-fluorouracil, Doxorubicin, Mustine, vincristine, and Procarba. Procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, cisplatin, Epirubicin, cisful Includes cisplatin, capecitabine, oxaliplatin, etc.

Cancer diseases defined at this hospital include leukemia, lymphoma, myeloma, myelodysplastic syndrome, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer (=colon cancer), rectal cancer, anal cancer, hepatocellular liver cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, and lung cancer (non-small cell cancer). genital lung cancer, small cell lung cancer), thymic cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, ovarian cancer, cervical cancer, sarcoma, gastrointestinal stromal tumor (GIST), cancer of unknown primary site, mesothelioma, melanoma, This includes neuroendocrine tumors, skin cancer, and blood cancer.

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

The extracts of the present invention can be obtained by the following production method.

By way of example, the present invention will be described in detail below.

The galangal extract of the present invention can be prepared as follows. After washing and chopping the dried steel, a solvent selected from water including purified water, lower alcohols with 1 to 4 carbon atoms such as methanol, ethanol, butanol, or mixed solvents thereof, preferably water or a mixed solvent with water, ethanol, and alcohol. , more preferably, water or a mixed solvent of water and alcohol or ethanol, more preferably 30 to 100% ethanol or alcohol, mixed several times and then heated to 30° C. at a temperature of 30° C. to 150° C., preferably 50° C. to 100° C. Obtained by repeating ultrasonic extraction, hot water extraction, room temperature extraction, or reflux extraction, preferably reflux cooling extraction, about 1 to 20 times, preferably 2 to 10 times, for minutes to 48 hours, preferably 24 hours to 36 hours. The crude extract of the present invention can be obtained by filtering, concentrating under reduced pressure, and drying the extract.

In addition, the polar solvent or non-polar solvent soluble extract of the present invention is prepared by adding water in a volume (v/w%) of about 0.0005 to 500 times the weight of the crude extract obtained above, preferably 0.05 to 5 times the volume (v/w%), then n-hexane, By performing a conventional fractionation process using methylene chloride, ethyl acetate, and butanol, non-polar solvent soluble extract fractions soluble in non-polar solvents such as n-hexane, methylene chloride, and ethyl acetate, and polar solvents soluble in polar solvents such as butanol and water. Usable extract fractions can be obtained separately.

Compounds of the present invention can be prepared as follows. For example, the crude extract of Yanggang obtained above is dispersed in water and the non-polar substances are fractionated with hexane or ethyl ether. Subsequently, the ethyl acetate soluble fraction obtained by fractionating the water layer with ethyl acetate was selectively purified using chromatography such as silica gel open column chromatography, flash column chromatography, RP C18 column chromatography, or Diaion HP-20 column chromatography. This can be repeated several times to purify and obtain each compound of the present invention.

The present inventors conducted an experiment (1) on the effect on myoblast differentiation (Experimental Examples 1 and 5) of the galangal extract and compounds obtained by the above production method to obtain cylinder-shaped cells by treating the samples. Differentiation into multinucleated myotubes was induced in a concentration-dependent manner, and the number of multinucleated myotubes was confirmed to increase. (2) Mechanism study of p38 MAPK signaling system for promoting myoblast differentiation (Experimental Example 2); (3) Through a muscle loss inhibition experiment of the galangal extract and compounds isolated therefrom in an in vitro muscle loss model (Experimental Examples 3, 4, and 6)), the samples of the present invention differentiated into muscle cells. It was confirmed that galangal extract and galangal-derived compounds can be used as a treatment or adjuvant for skeletal muscle disease by confirming that it not only promotes muscle loss in a muscle damage model, but that the composition can be used as a pharmaceutical for the prevention and treatment of skeletal muscle disease. It was confirmed to be useful as a composition or health functional food.

Therefore, the present invention provides a pharmaceutical composition or health functional food for the prevention and treatment of skeletal muscle-related diseases, containing as an active ingredient the galangal extract obtained by the above production method or a compound isolated therefrom.

The compounds of the present invention can be prepared into pharmaceutically acceptable salts and solvates according to methods common in the art.

As a pharmaceutically acceptable salt, an acid addition salt formed by a free acid is useful. Acid addition salts are prepared by conventional methods, for example, by dissolving the compound in an excess of aqueous acid and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone, or acetonitrile. Equimolar amounts of the compound and an acid or alcohol (e.g., glycol monomethyl ether) in water can be heated and the mixture then evaporated to dryness, or the precipitated salt can be filtered off with suction.

At this time, organic acids and inorganic acids can be used as free acids. Hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. can be used as inorganic acids, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, and trifluoroacetic acid can be used as organic acids. , citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid. (gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, and hydroiodic acid can be used.

Additionally, a pharmaceutically acceptable metal salt can be prepared using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate. At this time, it is particularly pharmaceutically suitable to prepare sodium, potassium or calcium salts as metal salts, and the corresponding silver salts are obtained by reacting an alkali metal or alkaline earth metal salt with an appropriate silver salt (eg, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the present invention, unless otherwise indicated, include salts of acidic or basic groups that may be present in the compounds of the present invention. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate and dihydrogen. There are phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate), and p -toluenesulfonate (tosylate) salts, and methods or processes for producing salts known in the art. It can be manufactured through.

The composition of the present invention contains the extract or compound in an amount of 0.01 to 99% by weight based on the total weight of the composition.

However, the composition as described above is not necessarily limited to this and may vary depending on the patient's condition and the type and progress of the disease.

A composition containing the extract or compound of the present invention may further include appropriate carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

The composition containing the extract according to the present invention is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, and sterile injection solutions according to conventional methods. Carriers, excipients, and diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, and calcium. Silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient in the extract, at least cotton, starch, calcium carbonate, and sucrose. Alternatively, it is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The preferred dosage of the extract or compound of the present invention varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. However, for a desirable effect, the extract is preferably administered at 0.01 mg/kg to 10 g/kg per day, preferably at 1 mg/kg to 1 g/kg. Administration may be administered once a day, or may be administered in several divided doses. Therefore, the above dosage does not limit the scope of the present invention in any way.

The composition of the present invention can be administered to mammals such as mice, live mice, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal, or intravenous.

The present invention also provides a therapeutic agent for muscular dystrophy or cachexia containing the above-mentioned galangal extract or a compound isolated therefrom as an active ingredient, and such therapeutic agent is intended to improve weight change or appetite recovery in combination therapy for muscular dystrophy or cachexia. In treatment, combination therapy using the composition of the present invention is provided rather than single administration of anticancer drugs.

The present invention also provides a treatment for muscular dystrophy or cachexia caused by cancer, which contains the above galangal extract or a compound isolated therefrom in combination with an existing anticancer agent as an active ingredient.

The present invention also provides an adjuvant anticancer treatment for skeletal muscle-related diseases caused by cancer, which uses the above galangal extract or a compound isolated therefrom in combination with an existing anticancer agent as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of skeletal muscle-related diseases, containing galangal extract or a compound isolated therefrom as an active ingredient.

In addition, the present invention provides a health functional food for the prevention and improvement of skeletal muscle-related diseases containing as an active ingredient a combination of galangal extract or a compound isolated therefrom and a conventional anticancer agent.

“Health functional food” as defined herein refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with Act No. 6727 on Health Functional Food. “Functional” refers to food that is useful for the human body. It means ingestion for the purpose of controlling nutrients for structure and function or obtaining useful health effects such as physiological effects.

The health functional food for preventing and improving skeletal muscle-related diseases of the present invention contains the extract or compound at a weight percentage of 0.01 to 95%, preferably 1 to 80%, based on the total weight of the composition.

Moreover, the composition of the present invention may be a health functional food or health supplement for the purpose of treating and improving skeletal muscle-related diseases.

In addition, for the purpose of preventing and improving skeletal muscle-related diseases, health functional foods in the form of pharmaceutical dosage forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions, and syrups, or in the form of tea bags, leached teas, and health drinks, etc. It can be manufactured and processed.

In addition, the present invention provides a health supplement for preventing and improving skeletal muscle-related diseases, containing galangal extract or a compound isolated therefrom as an active ingredient.

In addition, the present invention provides a health supplement for the prevention and improvement of skeletal muscle-related diseases containing as an active ingredient a combination of galangal extract or a compound isolated therefrom and a conventional anticancer agent.

The health functional or health supplement beverage composition of the present invention has no particular restrictions on other ingredients other than containing the above extract as an essential ingredient in the indicated ratio, and contains various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. can do. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract (e.g. rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. there is. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the composition of the present invention.

In addition, the present invention provides a food or food additive for preventing and improving skeletal muscle-related diseases containing a galangal extract or a compound isolated therefrom as an active ingredient.

In addition, the present invention provides a food or food additive for preventing and improving skeletal muscle-related diseases, containing as an active ingredient a combination of galangal extract or a compound isolated therefrom and a conventional anticancer agent.

When using the extract or compound according to the present invention as a food additive, the extract can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, These include alcoholic beverages and vitamin complexes, and include all health foods in the conventional sense.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the compositions of the present invention may contain pulp for the production of natural fruit juice and fruit juice beverages and vegetable beverages. These ingredients can be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Additionally, the extract of the present invention can be added to food or beverages for the purpose of preventing the target disease. At this time, the amount of the extract in the food or beverage can be added at 0.01 to 15% by weight of the total weight of the food, and the health drink composition can be added at a rate of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml. You can.

Through (1) an effect experiment (Experimental Examples 1 and 5) on myoblast differentiation using the galangal extract and compounds of the present invention, cylinder-shaped multinucleated myotube cells (multinucleated) were obtained by treatment of the samples. It was confirmed that differentiation into myotubes was induced in a concentration-dependent manner, and the number of multinucleated myotubes increased. (2) Mechanism study of the p38 MAPK signaling system for promoting myoblast differentiation (Experimental Example 2); (3) Through experiments on the inhibition of muscle loss with the galangal extract and the compounds isolated therefrom in an in vitro model of muscle loss (Experimental Examples 3, 4, and 6), it was found that the samples of the present invention promoted myogenic cell differentiation and myotubes. It has been confirmed to have a cytoprotective effect, preventing atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle stiffness, amyotrophic axonal sclerosis, myasthenia gravis, cachexia, and sarcopenia. ), etc. By confirming that it can be used as a treatment or supplement for skeletal muscle diseases, it was confirmed that the composition is useful as a pharmaceutical composition for preventing and treating muscle diseases, health functional foods, and health supplements.

Figure 1 is a diagram showing the effect of the galangal extract of the present invention on MHC, myoD, and myogenin expression;
Figure 2 is a diagram showing the change in the number of MHC positive-cylinder-shaped multinucleated myotube cells by the galangal extract of the present invention;
Figure 3 is a diagram showing the effect of the galangal extract of the present invention on the activation of the p38 MAP kinase (kinase) signaling system;
Figure 4 is a diagram showing the effect of the extract of the present invention on MHC or MuRF1 expression in a model in which muscle loss was induced by treating myotube cells differentiated by treatment with the galangal extract of the present invention with dexamethasone;
Figure 5 is a diagram showing the effect of galangal extract on the number of MHC-positive cylindrical multinucleated myotube cells in a muscle loss cell model;
Figure 6 is a diagram showing the effect of the compounds derived from the extract of the present invention on MHC expression in a model in which muscle loss was induced by treating dexamethasone in myotube cells differentiated by treatment with the compounds derived from the galangal extract of the present invention;
Figure 7 is a diagram showing the effect of Compound 2 of the present invention on MHC, myoD, and myogenin expression;
Figure 8 is a diagram showing the change in the number of MHC positive-cylinder-shaped multinucleated myotube cells by compound 2 of the present invention;
Figure 9 is a diagram showing the effect of the extract of the present invention on MHC or MuRF1 expression in a model in which muscle loss was induced by treating myotube cells differentiated by treatment with compound 2 of the present invention with dexamethasone;
Figure 10 is a diagram showing the effect of Compound 2 on the number of MHC-positive cylindrical multinucleated myotube cells in a muscle loss cell model.

Hereinafter, the present invention will be described in detail through the following reference examples and experimental examples.

However, the following reference examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following reference examples and experimental examples.

Example 1. Preparation of Yangjiang crude extract

50 g of dried Yang Kang (Alpinia officinarum: Rhizome, Jinheung Building Materials Pharmaceutical Co., Ltd.) was extracted three times by refluxing and cooling with 80% ethanol for 12 hours, and then concentrated under reduced pressure to produce the crude alcohol extract of Yang Kang (8.60 g, hereinafter referred to as “AOE”). got it

Example 2. Preparation example of galangal fraction

8 g of the 80% ethanol extract of Yanggang obtained in Example 1 was suspended by adding 100 mL of purified water, and the suspension was fractionated by adding 100 mL of n-hexane. The remaining water suspension was fractionated by adding 100 mL of ethyl acetate, and each was dried under reduced pressure to obtain n- 1.4 g and 2.5 g of the hexane soluble fraction (hereinafter referred to as AOH) and ethyl acetate soluble fraction (hereinafter referred to as AOEA) were obtained and freeze-dried.

Example 3. Isolation of active compounds from galangal extract

The ethyl acetate soluble fraction (2.50 g) of Yanggang was subjected to silica gel column chromatography (70-230 mesh, 190 g) under the conditions of Hexane: EtOAc (100:1 → 2:1) and divided into 10 fractions (F1 - F10). The F4 fraction (56 mg) was subjected to silica gel column chromatography using Hexane:EtOAc (30:1) to separate compound 4 (14 mg), and the F7 fraction (29 mg) was subjected to CH ₂ Cl ₂ :MeOH (200:1). Compound 7 (15 mg) was separated by silica gel column chromatography under the conditions of 1). The F8 fraction (640 mg) was subjected to silica gel column chromatography under the conditions of CH ₂ Cl ₂ :MeOH (100:0 → 200:1) to obtain compound 1 (32 mg), compound 5 (46 mg), and compound 8 ( 8 mg) were each purified, and the F9 fraction (446 mg) was subjected to silica gel column chromatography under the conditions of CHCl ₃ :MeOH (80:1) to obtain compound 2 (270 mg), compound 3 (5 mg), and compound 6 (76 mg) were purified from each other.

Compound 1 (brown crystal, Chem. Pharm. Bull. 1981, 29: 2383-2385), Compound 2 (brown crystal, Chem. Pharm. Bull. 1981, 29: 2383-2385), Compound 3 (brown crystal, Chem. Pharm. Bull. 1985, 33, 4889-4893), Compound 4 (brown crystal, Arch. Pharmacal Res. 2100, 34: 1289-1296), Compound 5 (brown oil, Chem. Pharm. Bull. 1985, 33: 4889). -4893), Compound 6 (brown crystal, Chem. Pharm. Bull. 1982, 30: 2279-2282), Compound 7 (brown crystal, Planta Med. 2008, 74: 427-431), Compound 8 (yellow crystal, Magn. Reson. Chem. 2001, 39, 374-380) were confirmed through comparative analysis based on the physical properties described in previously known references.

Compound 1: 7-(4-hydroxy-3-methoxyphenyl)-1-phenylhept-4-en-3-one [7-(4-hydroxy-3-methoxyphenyl)-1-phenylhept-4-en -3-one] HRFABMS m/z 311.1773 (calculated for C ₂₀ H ₂₃ O ₃ , 311.1647) 1H-NMR (CDCl ₃ , 400 MHz): δ 2.49 (2H, td, 7.6Hz, H-6), 2.70 ( 2H, t, 7.6Hz, H-7), 2.84 (2H, m, H-2), 2.93 (2H, t, 8.4Hz, H-1), 3.86 (3H, s, OCH3), 5.49 (1H, s, OH), 6.11 (1H, dt, 16Hz, 1.6Hz, H-4), 6.65 (1H, S, H-2"), 6.67 (1H, dd, 4Hz, 2Hz, H-6"), 6.83 (2H, m, H-5, H-5"), 7.19 (2H, t, 8Hz, H-2', H-6'), 7.20 (2H, t, 8Hz, H-4'), 7.28 ( 2H, t, 8Hz, H-3', H-5').13C-NMR (CDCl3, 100 MHz): δ 30.39 (C-1), 34.46 (C-7), 34.77 (C-6), 42.06 (C-2), 56.19 (OCH3), 111.19 (C-2"), 114.65 (C-5"), 121.22 (C-6"), 126.39 (C-4'), 128.65 (C-2', 6'), 128.78 (C-3', 5'), 130.98 (C-4), 132.90 (C-1"), 141.54 (C-1'), 144.32 (C-4"), 146.66 (C- 5), 146.75 (C-3"), 199.71 (C-3).

Compound 2: 5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenylheptan-3-one [5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1- phenylheptan-3-one] HRFABMS m/z 329.1652 (calculated for C ₂₀ H ₂₅ O ₄ , 329.1753) ¹ H-NMR (CDCl ₃ , 400 MHz): δ 1.59~1.82 (2H, m, H-6), 2.55 (2H, m, H-4), 2.40~2.76 (2H, m, H-7), 2.75 (2H, t, 7.6Hz, H-2), 2.90 (2H, t, 7.6Hz, H-1) , 3.09 (1H, s, 5-OH), 3.87 (3H, s, 3"-OCH ₃ ), 4.04 (1H, m, 4.0Hz, H-5), 5.53 (1H, s, 4"-OH) , 6.67 (1H, d, 8.0Hz, H-6"), 6.70 (1H, s, H-2"), 6.83 (1H, d, 8.0 Hz, H-5"), 7.18 (2H, t, 8.0 Hz, H-2', H-6'), 7.20 (1H, t, 8.0Hz, H-4'), 7.28 (2H, t ^, 8Hz, H-3', H-5'). NMR (CDCl ₃ , 100 MHz): δ 29.12 (C-1), 31.06 (C-7), 37.98 (C-6), 44.66 (C-2), 48.90 (C-4), 55.50 (3"- OCH ₃ ), 66.49 (C-5), 110.70 (C-2"), 113.90 (C-5"), 120.55 (C-6"), 125.87 (C-4'), 127.90 (C-2', 6'), 128.20 (C-3', 5'), 133.34 (C-1"), 140.28 (C-1'), 143.36 (C-4"), 146.04 (C-3"), 210.82 (C -3).

Compound 3: 5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)heptan-3-one [5-Hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)heptan -3-one] C ₂₁ H ₂₈ O ₆ (mw 374.43) ¹ H-NMR (CDCl ₃ , 400 MHz): δ 1.80 (2H, m, H-4), 2.52~2.84 (8H , m, H-1, H-2, H-6, H-7), 3.08 (1H, s, 5-OH), 3.90 (3H, s, OCH ₃ ), 4.07 (1H, m, H-5), 5.49 (1H, s, 4′-OH), 5.51 (1H, s, 4″-OH), 6.60 (2H, d, H-6′, H -6″), 6.62 (2H, s, H-2′, H-2″), 6.87 (2H, m, H-5′, H-5″). ¹³ C-NMR (CDCl ₃ , 100 MHz): δ 29.26 (C-7), 31.41 (C-1), 38.32 (C-6), 45.40 (C-2), 49.33 (C-4), 55.86 ( -OCH ₃ ), 66.83 (C-5), 110.91 (C-2′), 111.02 (C-2″), 114.21 (C-5″), 114.35 (C-5′), 120.70 (C-6′) , C-6″), 133.68 (C-1’, C-1″), 143.70 (C-4′, C-4″), 146.41 (C-3′, C-3″), 211.42 (C- 3).

Compound 4: 1,7-diphenylhept-4-en-3-one [1,7-diphenylhept-4-en-3-one]

HRFABMS m/z 265.1584 (calculated for C ₁₉ H ₂₁ O, 265.1592) ¹ H-NMR (CDCl ₃ , 400 MHz): δ 2.61 (2H, tdd, 8.0Hz, 8.0Hz, 1.6Hz, H-6), 2.85 (2H, t, 8.0Hz, H-7), 2.94 (2H, m, H-2), 3.01 (2H, t, 8.4Hz, H-1), 6.20 (1H, dt, 15.6Hz, 1.6Hz, H-4), 6.92 (1H, dt, 15.6Hz, 8.0Hz, H-5), 7.22~7.32 (6H, m, H-2', H-4', H-6', H-2", H-4", H-6"), 7.34~7.40 (4H, m, H-3', H-5', H-3", H-5") ¹³ C-NMR (CDCl ₃ , 100 MHz). ): δ 30.39 (C-1), 34.42 (C-6), 34.70 (C-7), 42.01 (C-2), 126.37 (C-4'), 126.53 (C-4"), 128.62 (C -2', 6'), 128.65 (C-2", 6"), 128.77 (C-3', 5'), 128.81 (C-3", 5"), 131.01 (C-4), 140.96 ( C-1"), 141.53 (C-1'), 146.59 (C-5), 199.71 (C-3).

Compound 5: 1,7-diphenyl-5-hydroxy-3-heptanone [1,7-diphenyl-5-hydroxy-3-heptanone]

HRFABMS m/z 283.1770 (calculated for C ₁₉ H ₂₃ O ₂ , 283.1698) ¹ H-NMR (CDCl ₃ , 400 MHz): δ 1.60~1.80 (2H, m, H-6), 2.53 (2H, m, H -4), 2.71~2.81 (2H, m, H-7), 2.73 (2H, t, 7.6Hz, H-2), 2.88 (2H, t, 7.6Hz, H-1), 3.03 (1H, s) , OH), 4.03 (1H, m, 4Hz, H-5), 7.10~7.28 (10H, m, Ring-H). ¹³ C-NMR (CDCl ₃ , 100 MHz): δ 29.47 (C-1), 31.71 (C-7), 37.99 (C-6), 44.99 (C-2), 49.24 (C-4), 66.83 ( C-5), 125.86 (C-4'), 126.21 (C-4"), 128.24 (C-2', 6'), 128.39 (C-2", 6"), 128.44 (C-3', 5'), 128.53 (C-3", 5"), 140.63 (C-1'), 141.78 (C-1"), 211.08 (C-3).

Compound 6: 5-hydroxy-7-(4-hydroxyphenyl)-1-phenyl-3-heptanone [5-hydroxy-7-(4-hydroxyphenyl)-1-phenyl-3-heptanone] HRFABMS m/ z 299.1720 (calculated for C ₁₉ H ₂₃ O ₃ , 299.1647) ¹ H-NMR (CDCl ₃ , 400 MHz): δ 1.56~1.8 (2H, m, H-6), 2.54 (2H, m, H-4) , 2.51~2.83 (2H, m, H-7), 2.75 (2H, t, 7.6Hz, H-2), 2.89 (2H, t, 7.6Hz, H-1), 3.05 (1H, s, 5- OH), 4.05 (1H, m, 4Hz, H-5), 5.19 (1H, s, 4"-OH), 6.74 (2H, d, 8.4Hz, H-3", 5"), 7.03 (2H, d, 8.4Hz, H-2", 6"), 7.17 (2H, t, 7.6Hz, H-2', 6), 7.19 (1H, t, 7.6Hz, H-4'), 7.28 (2H, t, 7.6 Hz, H-3', H-5') ¹³ C-NMR (CDCl ₃ , 100 MHz): δ 29.47 (C-1), 30.75 (C-7), 38.16 (C-6), 44.99 (C-2), 49.20 (C-4), 66.88 (C-5), 115.23 (C-3", 5"), 126.22 (C-4'), 128.24 (C-2", 6") , 128.53 (C-3', 5'), 129.49 (C-2", 6"), 133.70 (C-1"), 140.60 (C-1'), 153.80 (C-4"), 211.08 (C -3).

Compound 7: 1,7-diphenyl-5-methoxy-3-heptanone [1,7-diphenyl-5-methoxy-3-heptanone]

HRFABMS m/z 297.1809 (calculated for C ₂₀ H ₂₅ O ₂ , 297.1855) ¹ H-NMR (CDCl ₃ , 400 MHz): δ 1.61~1.84 (2H, m, H-6), 2.53 (2H, m, H -4), 2.58~2.82 (2H, m, H-7), 2.73 (2H, t, 7.6Hz, H-2), 2.88 (2H, t, 7.6Hz, H-1), 3.49 (3H, s) , OCH ₃ ), 4.10 (1H, m, 4Hz, H-5), 7.10~7.28 (10H, m, Ring-H). ¹³ C-NMR (CDCl ₃ , 100 MHz): δ 29.45 (C-1), 31.68 (C-7), 37.99 (C-6), 44.97 (C-2), 49.25 (C-4), 50.78 ( OCH ₃ ), 66.83 (C-5), 125.84 (C-4'), 126.19 (C-4"), 128.23 (C-2', 6'), 128.37 (C-3', 5'), 128.41 (C-2", 6"), 128.51 (C-3", 5"), 140.61 (C-1'), 141.75 (C-1"), 211.11 (C-3).

Compound 8: 3,5,7-trihydroxy-2-phenyl-chromen-4-one (galangin) 3,5,7-trihydroxy-2-phenyl-chromen-4-one (galangin); C ₁₅ H ₁₀ O ₅ (mw 270.05) ¹ H-NMR (Acetone-d ₃ , 400 MHz): δ 2.1 (3H, s, -OH), 6.3 (1H, d, H-6), 6.5 (1H, d, H-8), 7.5 (1H, d, H-4′), 7.6 (2H, d, H-3′H-5′), 8.3 (2H, d, H-2′H-6′) , ¹³ C-NMR (Acetone-d ₃ , 100 MHz): δ 94.6 (C-8), 99.2 (C-6), 104.3 (C-10), 128.5 (C-2′, C-6′), 129.4 (C-3′, C-5′), 130.9 (d, C-4′), 132.1 (s, C-1′), 137.9 (s, C-3), 146.1 (s, C-2) 158.1 (s, C-9), 162.2 (s, C-5), 165.2 (s, C-7), 176.9 (s, C-4),

Experimental Example 1. Evaluation of the efficacy of galangal extract to promote myoblast differentiation

In order to confirm the effect of the galangal extract of the above example on the differentiation (myogenic effect) of myogenic cells, an experiment was performed by applying the method described in the existing literature as follows. ((11. Chem. Biol. Interact. 2016, 248, 60).

1-1. experimental process

C2C12 cells (CRL-1771, ATCC), a mouse-derived myogenic cell line, were differentiated into myotube cells by treating extracts 1, 10, and 100 ng/ml, respectively. Cell lysates of differentiated cells were obtained and the protein expression levels of MHC, myoD, and myogenin were evaluated through Western blotting (Figure 1), and mouse anti-MHC and fluorescent substance-conjugated anti-mouse antibodies were used. (anti-mouse IgG2b Alexa-fluor 568) immunostaining was performed to evaluate changes in MHC expression in myotube cells due to the extract (Figure 2) ( Chem. Biol. Interact. 2016, 248, 60).

Specifically, C2C12 myogenic cells (CRL-1771, ATCC) were treated with galangal extract (1, 10, 100 ng/mL) and grown in differentiation medium (2% horse serum-containing DMEM, Cat ^# 11965-084, Differentiation was induced by treatment on Gibco for 3 days.

1-1-1. Western blot analysis

To ^perform Western blot analysis, approximately 3 (lysis buffer, 25mM Tris-HCl (pH 7.5), 100mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail (Cat# 04-906-845 -001, Roche) protein extract was obtained, and 20 μg of the protein extract was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. -376157, Santa Cruz), anti-myoD (sc-32758, Santa Cruz), or anti-myogenin (sc-12732, Santa Cruz) were bound at 4°C for 12 hours, followed by horseradish peroxidase-linked anti-mouse (goat). After binding anti-mouse IgG-HRP, ADI-SAN-100J, Enzo Life Science) or anti-rabbit (goat anti-rabbit IgG-HRP, ADI-SAN-300J, Enzo Life Science) secondary antibody, chemiluminescence The protein amount was analyzed at this time, pan-cadherin (anti-pan-cadherin antibody, Cat# 3678, Sigma) was used as a loading control.

1-1-2. Immunofluorescence staining

Myogenic cells were differentiated by treating differentiation medium with each sample added in the same manner as above, and immunofluorescence staining was performed.

Each differentiation medium was removed, washed twice with phosphate-buffered saline, and fixed with 4% paraformaldehyde (0141, BBC Biochemical) for 20 minutes. It was again washed twice with phosphate-buffered saline and treated with 0.1% tritonX-100 (2315025, Sigma) for 20 minutes. After washing twice with phosphate-buffered saline and blocking with 5% horse serum solution (16050122, Gibco), mouse anti-MHC (MAB4470, R&D systems) was added and reacted at 4°C for 12 hours.

After reaction, wash with phosphate-buffered saline at least three times, stain MHC using Alexa Flouor 568-conjugated secondary anti-mouse (A-21144, MicoProbes), and stain intracellular nuclei using DAPI (D9542, Sigma). did. Immunofluorescence results of MHC-positive myotubes visualized MHC expression in red and DAPI-labeled nuclei in blue.

1-2. Experiment result (Table 1-2, Figure 1-2)

In Figure 1, the effect of galangal extract on MHC, myoD, and myogenin expression was measured. The extract was treated with the myoblast differentiation medium at a concentration of 1, 10, and 100 ng/mL, differentiated into myotube fibers for 3 days, and cell lysates were obtained to determine protein expression of MHC, myoD, and myogenin, which are differentiation markers for myotube fibers. was analyzed by Western blotting analysis.

As a result of the analysis, when the expression of MHC, myoD, and myogenin in the control cells was set to 1, the expression level of each marker increased as shown in Table 1 by treatment with the galangal extract (Table 1).

In Figure 2, the muscle differentiation activity of the extract was measured by immunofluorescence staining using anti-MHC antibodies and DAPI. Increased red-fluorescence indicates that the extract promotes MHC expression in C2C12 cells, and by visualizing the nuclei through DAPI counter staining, they are multinucleated and have a cylindrical shape where MHC is expressed. It was confirmed that the number of (cylinder-shaped) myotube cells increased depending on the concentration of the extract (Table 2).

In the above experiment, it was found that galangal extract increased the expression of myocyte differentiation markers in myotube cells and the number of cylinder-shaped multinucleated myotubes in a concentration-dependent manner, promoting muscle cell differentiation. was proven (Table 1-2 and Figure 1-2).

galangal extract [ng/mL] 0 One 10 100 MHC
/pan-cadherin expression (fold) 1.0 ^a 1.3 ^b 1.3 ^c 1.7 ^d myoD
/pan-cadherin expression (fold) 1.0 ^ab 1.1 ^a 3.9 ^b 5.2 ^c myogenin
/pan-cadherin expression (fold) 1.0 ^ab 1.1 ^b 1.0 ^a 1.2 ^{c a,b,c} : Different letters indicate significance ( p < 0.01)

Increased activity of MHC, myoD and myogenin expression by galangal extract (Figure 1)

galangal extract [ng/mL] 0 One 10 100 Number (fold) of myotube fibers that are multinucleated with five or more nuclei and express MHC 1.0 ^a 2.0 ^b 2.0 ^b 2.2 ^{c a,b,c} : Different letters indicate significance ( p < 0.01)

Changes in the number of MHC positive-cylinder-shaped multinucleated myotube cells by galangal extract (Figure 2)

Experimental Example 2. Study on the mechanism of the p38 MAPK signaling system on the promotion of myogenic cell differentiation by Yangkang extract

The effect of the galangal extract of the above example on the p38-mitogen-activated protein kinase (p38 MAPK) signaling system mechanism during myogenic cell differentiation was confirmed.

The p38 MAPK signaling system is known to be a central mechanism of myoblast differentiation, and p38 MAPK induces the expression of myogenic factors by promoting dimerization of the transcription factor MyoD. ( Trends Cell Biol . 2006 , 16 , 36). Therefore, to measure the activation of p38 MAPK by galangal extract, an experiment was performed by applying the method described in the existing literature as follows. ( Mol. Biol. Cell. 2010 , 21, 2399; Trends Cell Biol . 2006 , 16 , 36).

2-1. experimental process

The present inventors treated myoblasts with extracts (1, 10, and 100 ng/mL), obtained lysates from differentiated myotubes on day 3 of differentiation, and analyzed the expression of phosphorylated p38 MAPK protein, which is the activated form of p38 MAPK. Levels were analyzed by Western blotting analysis.

For this purpose, the expression of phosphorylated p38 MAPK was measured using a primary rabbit-anti-phosphorylated p38 MAPK (Cat# 9211, Cell Signaling Technology) and an anti-rabbit secondary antibody (goat anti-rabbit IgG-HRP, sc-2004, SantaCruz). At this time, the loading control was the total expression level of p38 MAPK, and primary rabbit-anti-p38 MAPK (Cat# 9212, Cell Signaling Technology) was used to analyze this.

2-2. Experimental results (Table 3 and Figure 3)

As a result of confirming the effect of the extract on p38 MAPK activation by Western analysis, the p38 MAPK signaling system, which is important for myogenic cell differentiation, was further activated by the extract during the differentiation period, and this was proportional to the treatment concentration of the extract (Table 3).

The above experimental results indicate that p38 MAPK activation occurs when myoblast differentiation is promoted by the galangal extract.

galangal extract [ng/mL] 0 One 10 100 Phosphorylation-p38
/p38 expression level (fold) 1.0 ^a 1.9 ^b 2.4 ^c 2.8 ^{d a,b,c,d} : Different letters indicate significance ( p < 0.01)

p38 MAPK activity of galangal extract (Figure 3)

Experimental Example 3. Inhibitory muscle loss efficacy of galangal extract in an in vitro muscle loss model

In order to confirm the muscle loss inhibition effect of the galangal extract of the above example, an experiment was performed by applying the method described in the existing literature as follows. ( Int. J. Mol. Med. 2015, 36 , 29-42; Biomed. Pharm. 2017 , 95 , 1486)

An in vitro model of muscle loss was induced by adding dexamethasone (200 μM), a synthetic glutinoid, to myotube cells differentiated for 3 days by treating the extract of the above example ( Int. J. Mol. Med. 2015, 36 , 29-42).

To determine whether the extract inhibits muscle protein loss, we used Western blotting analysis to determine how it affects the expression of MHC, a myotube cell marker protein, and the protein expression of MuRF1, a muscle protein degrading enzyme that induces muscle loss (Figure 4 ), it was re-confirmed using immunofluorescence staining whether the loss of MHC protein in myotube cells caused by dexamethasone was inhibited by the extract (Figure 5).

3-1. experimental process

3-1-1. Western blot analysis

For Western blot analysis, ^{approximately} 3 Differentiated daily. Differentiated myotube cells were treated with 200 μM dexamethasone (D4902, Sigma) for 24 hours.

Cell lysis buffer (25mM Tris-HCl (pH 7.5), 100mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail was added to the treated cells. (Calbiochem, Darmstadt, Germany) to obtain a protein extract, and 20 μg of the protein extract was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. (sc-376157, Santa Cruz), or primary mouse anti-MuRF1 (sc-398608, Santa Cruz) was bound at 4°C for 12 hours, followed by horseradish peroxidase-linked anti-mouse (goat anti-mouse IgG-HRP, After binding the secondary antibody (ADI-SAN-100J, Enzo Life Science), the MHC and MuRF1 protein amounts were analyzed by chemiluminescence, using pan-cadherin (Cat# 3678, Sigma) as a loading control. did.

3-1-2. Immunofluorescence staining

Immunofluorescence staining was performed on in vitro model cells in which myotube cells differentiated at each extract concentration were damaged with dexamethasone in the same manner as above.

Each medium was removed, washed twice with phosphate-buffered saline, and fixed with 4% paraformaldehyde (0141, BBC Biochemical) for 20 minutes. It was again washed twice with phosphate-buffered saline and treated with 0.1% tritonX-100 (2315025, Sigma) for 20 minutes. After washing twice with phosphate-buffered saline and blocking with 5% horse serum solution (16050122, Gibco), mouse anti-MHC (MAB4470, R&D systems) was added and reacted at 4°C for 12 hours.

After the reaction, the cells were washed with phosphate-buffered saline at least three times, and MHC expression was analyzed using Alexa Flouor 568-conjugated secondary anti-mouse (A-21144, MicoProbes) and DAPI (D9542, Sigma). Immunofluorescence results of MHC-positive myotubes were visualized in red, and DAPI-labeled nuclei were visualized in blue.

3-2. Experiment results (Table 4-5, Figure 4-5)

As a result of analyzing MHC protein expression in each cell group using Western blotting analysis, when MHC expression in control (NC) myotube cells not treated with dexamethasone was set to 1, in the myotube cell group treated with dexamethasone to induce muscle loss, MHC expression was decreased by 0.3-fold. Compared to the muscle-loss myotube cell group, it was observed that in the cell group treated with the extract, MHC expression was restored to the control level depending on the concentration of the extract.

In addition, compared to the MuRF1 expression in the control (NC) myotube cells that were not treated with dexamethasone at 1, the expression of MuRF1 in the muscle loss cell group treated with dexamethasone increased 1.7-fold, and in the myotube cell group treated with 100 nM of the extract, the Compared to MuRF1 expression in the myotube cell population, a muscle loss model, it was barely expressed (Table 4, Figure 4).

As shown in Figure 5, in order to confirm the ability to inhibit muscle protein degradation reduced by dexamethasone, MHC expression was evaluated at the level of red fluorescence in the extract and dexamethasone-added myotube cells, and DAPI staining showed cylinder-shaped cells. ) The formation of multinucleated myotubes was evaluated. Treating differentiated myotube cells with dexamethasone increased the loss of myotube cells, but on the contrary, myotube cells obtained by treating the extract during differentiation suppressed the loss of multinucleated MHC-positive cells caused by dexamethasone ( Table 5 and Figure 5). It was demonstrated that muscle protection by the sample of the present invention occurred effectively and that muscle proteins were protected through inhibition of muscle proteolytic enzyme expression.

Myotube cell protective activity by galangal extract (Figure 4)

In dexamethasone-induced myotube cell loss, changes in the number of MHC positive-cylinder-shaped multinucleated myotube cells by galangal extract (Figure 5)

Experimental Example 4. Inhibitory muscle loss efficacy of extract-derived compounds in an in vitro model of muscle loss

Compounds derived from the galangal extract of the above example In order to confirm the inhibitory effect on muscle loss, an experiment was performed by applying the method described in the existing literature as follows. ( Int. J. Mol. Med. 2015, 36 , 29-42; Biomed. Pharm. 2017 , 95 , 1486)

Myotube cells treated with the compounds of the above example at a concentration of 10 nM were treated with dexamethasone to create an in vitro model of muscle loss ( Int. J. Mol. Med. 2015, 36 , 29-42) as follows. An experiment was conducted.

To analyze the effectiveness of compounds in suppressing muscle protein loss, Western blotting analysis was used to determine how the compounds affected MHC expression in myotubes (FIG. 6).

4-1. experimental process

Approximately ³ 0, 10 nM) were added and cultured for 3 days to differentiate.

To induce muscle loss ( in vitro) , myotube cells differentiated as above were treated with 200 μM dexamethasone (D4902, Sigma) for 24 hours. After preparing the cells by washing them with phosphate-buffered saline, the cells were added with lysis buffer (25mM Tris-HCl (pH 7.5), 100mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl). Protein extracts were obtained by treatment with sulfate (SDS), and protease inhibitor cocktail (Calbiochem, Darmstadt, Germany), and 20 μg of protein extracts were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and then transferred to polyvinylidene fluoride (PVDF) membranes. Primary mouse anti-MHC (sc-376157, Santa Cruz) was bound to this blot for 12 hours at 4 °C, followed by horseradish peroxidase-linked anti-mouse (goat anti-mouse IgG-HRP, ADI-SAN-100J). , Enzo Life Science), the MHC protein amount was analyzed by chemiluminescence. At this time, pan-cadherin (Cat# 3678, Sigma) was used as a loading control.

4-2. Experimental results (Table 6, Figure 6)

As a result of analyzing MHC protein expression in each cell group using Western blotting analysis, when MHC expression in control (NC) myotube cells not treated with dexamethasone was set to 1, in the myotube cell group treated with dexamethasone to induce muscle loss, MHC expression was decreased by 0.3-fold. When treated with Yanggang-derived compounds, MHC expression was restored to the control level of muscle-losing myotube cells, and in particular, compounds 1, 2, and 3 were observed to increase by 1.5-fold, 2.8-fold, and 2.0-fold, respectively, compared to the control group (Table 6, Figure 6).

It was confirmed that the compounds derived from the galangal extract of the present invention strongly inhibit muscle loss.

Myotube cell protective activity by extract-derived compounds (Figure 6)

Experimental Example 5 Evaluation of the efficacy of Compound 2 isolated from Yanggang extract to promote myoblast differentiation

In order to confirm the efficacy of Compound 2 isolated from the galangal extract of the above example in promoting cell differentiation (myoblast differentiation), an experiment was performed by applying the method described in the existing literature as follows. ( Chem. Biol. Interact. 2016, 248, 60).).

5-1. experimental process

In order to determine whether Compound 2 isolated from the galangal extract of the above example promotes myogenic effect, C2C12 cells, a mouse myogenic cell line, were treated with Compound 2 at concentrations of 1, 10, and 100 nM for 3 days and then differentiated. The lysate of the myotube cells was obtained and the protein expression level of MHC was evaluated through Western blotting analysis (Figure 7), and the protein expression level of MHC was assessed using mouse anti-MHC and anti-mouse antibody conjugated to a fluorescent substance (anti-mouse IgG2b Alexa- fluor 568) immunostaining was performed to evaluate the increase in cylinder-shaped multinucleated myotubes (FIG. 8) ( Chem. Biol. Interact. 2016, 248, 60).

Differentiation medium (2% horse serum-containing DMEM, Cat ^# 11965-084) supplemented with Compound 2 (1, 10, 100 nM) isolated from horsetail extract to C2C12 myogenic cells (Cat ^# CRL-1771, ATCC) , Gibco) for 3 days to induce differentiation.

5-1-1. Western blot analysis

For Western blot analysis, approximately ³ lysis buffer, 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail (Calbiochem, Darmstadt, Germany) protein extract 20 μg of protein extract was subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes with primary mouse anti-MHC (sc-376157, Santa Cruz) at 4°C for 12 hours. After binding, the horseradish peroxidase-linked anti-mouse secondary antibody (goat anti-mouse IgG-HRP, ADI-SAN-100J, Enzo Life Science) was reacted, and the protein amount was analyzed by chemiluminescence. Pan-cadherin (anti-pancadherin, Cat# 3678, Sigma) was used as a loading control.

5-1-2. Immunofluorescence staining

Myoblasts were differentiated in differentiation medium containing each concentration of Compound 2 in the same manner as above, and immunofluorescence staining was performed. Each differentiation medium was removed, washed twice with phosphate-buffered saline, and fixed with 4% paraformaldehyde (0141, BBC Biochemical) for 20 minutes. It was again washed twice with phosphate-buffered saline and treated with 0.1% tritonX-100 (2315025, Sigma) for 20 minutes. After washing twice with phosphate-buffered saline and blocking with 5% horse serum solution (16050122, Gibco), mouse anti-MHC (MAB4470, R&D systems) was added and reacted at 4°C for 12 hours. After the reaction, the cells were washed with phosphate-buffered saline at least three times, and MHC expression was analyzed using Alexa Flouor 568-conjugated secondary anti-mouse (A-21144, MicoProbes) and DAPI (D9542, Sigma). Immunofluorescence results of MHC-positive myotubes were visualized in red, and DAPI-labeled nuclei were visualized in blue.

5-2. Experiment result (Table 7-8, Figure 7-8)

In Figure 7, the effect of compound 2 on MHC expression was measured. Myoblast differentiation medium was treated with Compound 2 at concentrations of 1, 10, and 100 nM to differentiate into myotube fibers for 3 days. The cells were harvested, and the protein expression of MHC, a myoblast differentiation marker, was analyzed by Western blotting analysis.

As a result of the analysis, MHC expression increased in cells treated with Compound 2 compared to MHC expression in control cells, and peaked at a concentration of 1 nM of Compound 2 (Table 7).

In Figure 8, myoblast differentiation activity by compound 2 was measured by immunofluorescence staining using an anti-MHC antibody and DAPI. Increased red-fluorescence indicates that Compound 2 promotes MHC expression in C2C12 cells, and DAPI counter staining showed that cylinder-shaped myotube cells expressing MHC were multinucleated. It was confirmed that it was multinucleated.

In the above experiment, it was found that Compound 2 isolated from the galangal extract increased MHC expression and the number of cylinder-shaped multinucleated myotubes in myotube cells, demonstrating that it promoted muscle cell differentiation ( Table 7-8 and Figure 7-8).

compound 2 [nM] 0 One 10 100 MHC/pan-cadherin expression (fold) 1.0 ^a 3.6 ^b 2.6 ^c 2.6 ^{c a,b,c} : Different letters indicate significance ( p < 0.01)

MHC expression increasing activity by compound 2 (Figure 7)

compound 2 [10 nM] 0 One 10 100 Number of myotube fibers that are multinucleated with five or more nuclei and express MHC. 1.0 ^a 2.2 ^b 1.6 ^c 1.5 ^{c a,b,c} : Different letters indicate significance ( p < 0.01)

Change in the number of MHC positive-cylinder-shaped multinucleated myotube cells caused by compound 2 (Figure 8)

Experimental Example 6. Inhibitory muscle loss efficacy of compound 2 in an in vitro model of muscle loss

Compound 2 derived from the galangal extract of the above example In order to confirm the inhibitory effect on muscle loss, an experiment was performed by applying the method described in the existing literature as follows. ( Int. J. Mol. Med. 2015, 36 , 29-42; Biomed. Pharm. 2017 , 95 , 1486)

Myotube cells treated with Compound 2 of the above example at different concentrations were treated with dexamethasone to create an in vitro model of muscle loss ( Int. J. Mol. Med. 2015, 36 , 29-42) and conducted as follows. proceeded.

To analyze the efficacy of Compound 2 in suppressing muscle protein loss, Western blotting analysis was used to determine the effect on MHC expression in myotubes and the protein expression of MuRF1, a muscle protein degrading enzyme that induces muscle loss (Figure 9). Immunofluorescence staining was used to reconfirm whether the loss of MHC proteins in myotube cells caused by dexamethasone is inhibited by Compound 2 (FIG. 10).

6-1. experimental process

Approximately ³ , 1, 10, 100 nM) was added and cultured for 3 days to differentiate.

To induce muscle loss ( in vitro) , myotube cells differentiated as above were treated with 200 μM dexamethasone (D4902, Sigma) for 24 hours.

Cells treated with Compound 2 were prepared by washing with phosphate-buffered saline, and immunofluorescence staining was performed to confirm MHC expression (FIG. 10).

6-1-1. Western blot analysis

For Western blot analysis, ^{approximately} 3 I ordered it. After treating differentiated myotube cells with 200 μM dexamethasone (D4902, Sigma) for 24 hours, the cells were incubated with lysis buffer (25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% NP-40). , 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail (Calbiochem, Darmstadt, Germany) were used to obtain protein extracts, and 20 μg of protein extract was subjected to SDS-polyacrylamide gel electorphoresis (PAGE). , and transferred to polyvinylidene fluoride (PVDF) membranes. Primary mouse anti-MHC (sc-376157, Santa Cruz) or primary mouse anti-MuRF1 (sc-398608, Santa Cruz) was bound to this blot at 4°C for 12 hours. Then, horseradish peroxidase-linked anti-mouse (goat anti-mouse IgG-HRP, ADI-SAN-100J, Enzo Life Science) secondary antibody was coupled, and the amount of MHC and MuRF1 proteins was analyzed by chemiluminescence. pan-cadherin (Cat# 3678, Sigma) was used as a loading control.

6-1-2. Immunofluorescence staining

Myoblasts were differentiated in differentiation medium containing each sample in the same manner as above, and immunofluorescence staining was performed.

Each differentiation medium was removed, washed twice with phosphate-buffered saline, and fixed with 4% paraformaldehyde (0141, BBC Biochemical) for 20 minutes. It was again washed twice with phosphate-buffered saline and treated with 0.1% tritonX-100 (2315025, Sigma) for 20 minutes. After washing twice with phosphate-buffered saline and blocking with 5% horse serum solution (16050122, Gibco), mouse anti-MHC (MAB4470, R&D systems) was added and reacted at 4°C for 12 hours.

After the reaction, the cells were washed with phosphate-buffered saline at least three times, and MHC expression was analyzed using Alexa Flouor 568-conjugated secondary anti-mouse (A-21144, MicoProbes) and DAPI (D9542, Sigma). Immunofluorescence results of MHC-positive myotubes were visualized in red, and DAPI-labeled nuclei were visualized in blue.

6-2. Experiment results (Table 9-10, Figure 9-10)

As a result of analyzing MHC protein expression in each cell group using Western blotting analysis, when MHC expression in control (NC) myotube cells not treated with dexamethasone was set to 1, in the myotube cell group treated with dexamethasone to induce muscle loss, MHC expression was decreased by 0.8-fold. Compared to the muscle-loss myotube cell group, it was observed that MHC expression was restored to twice the control level in the cell group treated with Compound 2.

In addition, compared to MuRF1 expression in the control (NC) myotube cells not treated with dexamethasone, MuRF1 expression in the muscle wasting cell group treated with dexamethasone increased 7-fold, and in the myotube cell group treated with compound 2, it increased 7-fold in the dexamethasone-treated muscle. It decreased compared to MuRF1 expression in the myotube cell population, which is a loss model (Table 9, Figure 9).

As shown in Figure 10, in order to confirm the ability to inhibit muscle protein degradation reduced by dexamethasone, MHC expression was evaluated at the level of red fluorescence in myotube cells to which compound 2 and dexamethasone were added, and DAPI readout staining was performed on compound 2 treatment. The formation of cylinder-shaped multinucleated myotubes was evaluated. Treating differentiated myotube cells with dexamethasone increased the loss of myotube cells, but on the contrary, myotube cells obtained by treating compound 2 during differentiation suppressed the loss of multinucleated MHC-positive cells caused by dexamethasone. (Table 10 and Figure 10). It was demonstrated that muscle protection by Compound 2 of the present invention occurred effectively and that muscle proteins were protected through inhibition of muscle proteolytic enzyme expression.

Myotube cell protective activity by compound 2 (Figure 9)

In dexamethasone-induced myotube cell loss, change in the number of MHC positive-cylinder-shaped multinucleated myotube cells by compound 2 (FIG. 10)

Below, a preparation example of a composition containing the sample extract of the present invention is described, but the present invention is not intended to be limited, but merely explained in detail.

Formulation Example 1. Preparation of powder

Extract (AOE) ----------------------------------------- 20 mg

Lactose ------------------------------------------------- --100 mg

Talc ------------------------------------------------- --- 10mg

The above ingredients are mixed and filled into an airtight bubble to prepare a powder.

Formulation Example 2. Preparation of tablets

Compound 2 ------------------------------- 10 mg

Corn starch -------------------------------------------- 100 mg

Lactose ------------------------------------------------- - 100 mg

Magnesium stearate ------------------------------------- 2 mg

After mixing the above ingredients, tablets are manufactured by compressing them according to a typical tablet manufacturing method.

Formulation Example 3. Preparation of capsules

Fraction (AOEA) ----------------------------------------- 10 mg

Crystalline cellulose --------------------------------------- 3 mg

Lactose ------------------------------- 14.8 mg

Magnesium stearate --------------------------------- 0.2 mg

Capsules are prepared by mixing the above ingredients and filling them into gelatin capsules according to a typical capsule manufacturing method.

Formulation Example 4. Preparation of injections

Extract (AOE) ----------------------------------------------------------- 10 mg

Mannitol ------------------------------------------------ 180 mg

Sterile distilled water for injection -------------------- 2974 mg

Na ₂ HPO ₄ ,12H2O ---------------------------- 26 mg

It is prepared with the above ingredients per ampoule (2 ml) according to the usual manufacturing method for injections.

Formulation Example 5. Preparation of liquid formulation

Compound 2 ------------------------------------------------ 20 mg

Iseonghwadang ------------------------------------------------ - 10g

Mannitol ------------------------------------------------- ---5g

Purified water ------------------------------------------------- -- Appropriate amount

According to the usual liquid preparation method, add and dissolve each ingredient in purified water, add an appropriate amount of lemon flavor, mix the above ingredients, add purified water, adjust the total to 100 ml by adding purified water, and fill it in a brown bottle. Sterilize to prepare a liquid.

Formulation Example 6. Preparation of health food

Extract (AOE) ------------------------------------------1000 mg

Vitamin mixture -------------------------------------------- Appropriate amount

Vitamin A Acetate ------------------------------------- 70 ㎍

Vitamin E ----------------------------------------------- 1.0 ㎎

Vitamin B1 ---------------------------------------------- 0.13 mg

Vitamin B2 ------------------------------ 0.15 mg

Vitamin B6 ----------------------------------------------- 0.5 mg

Vitamin B12 ------------------------------- 0.2 ㎍

Vitamin C ------------------------------------------------ 10 mg

Biotin ------------------------------------------------- - 10 ㎍

Nicotinamide ------------------------------------------ 1.7 mg

Folic acid ------------------------------------------------- --- 50 ㎍

Calcium pantothenate ------------------------------------------ 0.5 mg

Mineral mixture -------------------------------------------- Appropriate amount

Ferrous sulfate ------------------------------- 1.75 ㎎

Zinc oxide ----------------------------------------------- 0.82 mg

Magnesium carbonate ------------------------------------------ 25.3 mg

Potassium Phosphate Monobasic ---------------------------------------------- 15 ㎎

Dibasic Calcium Phosphate --------------------------------------------- 55 ㎎

Potassium citrate ---------------------------------------------- 90 mg

Calcium carbonate ----------------------------------------------- 100 ㎎

Magnesium chloride ------------------------------------------ 24.8 mg

The composition ratio of the above vitamin and mineral mixture is a mixture of components relatively suitable for health food in a preferred embodiment, but the mixing ratio may be modified arbitrarily. The above ingredients are mixed according to a typical health food manufacturing method, and then , granules can be manufactured and used to manufacture health food compositions according to conventional methods.

Formulation example 7. Preparation of health drink

Compound 2 ----------------------------------------------- 1000 ㎎

Citric acid ------------------------------------------------- 1000 mg

oligosaccharide ------------------------------------------------- 100g

Plum concentrate ------------------------------------------------ - 2g

Taurine ------------------------------------------------- ----1g

Add purified water ------------------------------------ total 900 ml

After mixing the above ingredients according to a typical health drink manufacturing method, stirring and heating at 85° C. for about 1 hour, the resulting solution was filtered, obtained in a sterilized 2-liter container, sealed, sterilized, and refrigerated, followed by the present invention. It is used in the production of health drink compositions.

The composition ratio is a preferred embodiment of mixing ingredients that are relatively suitable for beverages of preference, but the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as demand class, demand country, and intended use.

Claims (27)

It contains as an active ingredient an extract of Alpinia officinarum rhizome soluble in a mixed solvent of water and alcohol or ethanol, and is used to treat diseases such as senile muscular atrophy or cancer-related muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, selected from the group consisting of amyotrophic axonal sclerosis, myasthenia gravis, cachexia, sarcopenia, and muscle loss. Pharmaceutical composition for preventing and treating skeletal muscle-related diseases.
delete delete delete delete delete delete delete delete delete delete delete delete delete Muscular atrophy, muscular dystrophy, muscle degeneration, muscular rigidity, and amyotrophic axonal sclerosis caused by cancer, containing as an active ingredient an extract of Alpinia officinarum rhizome soluble in a mixed solvent of water and alcohol or ethanol. , an adjuvant anti-cancer treatment for skeletal muscle-related diseases selected from the group consisting of myasthenia gravis, cachexia, sarcopenia, and muscle loss.
delete delete It contains as an active ingredient an extract of Alpinia officinarum rhizome soluble in a mixed solvent of water and alcohol or ethanol, and is used to treat diseases such as senile muscular atrophy or cancer-related muscular atrophy, muscular dystrophy, muscle degeneration, and muscular rigidity. A health functional food for preventing or improving skeletal muscle-related diseases selected from the group consisting of amyotrophic axonal sclerosis, myasthenia gravis, cachexia, senile sarcopenia, and muscle loss. The health functional food according to claim 18, wherein the health functional food is in the form of powder, granule, tablet, capsule, pill, suspension, emulsion, syrup, tea bag, leached tea, or health drink. delete delete It contains as an active ingredient an extract of Alpinia officinarum rhizome soluble in a mixed solvent of water and alcohol or ethanol, and is used to treat diseases such as senile muscular atrophy or cancer-related muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, A health supplement for preventing or improving skeletal muscle-related diseases selected from the group consisting of amyotrophic axonal sclerosis, myasthenia gravis, cachexia, senile sarcopenia, and muscle loss.
delete delete delete delete delete