KR102022279B1 - A composition comprising an extract of Angelica keiskei for treating and preventing muscle-related disorder - Google Patents

Abstract

The present invention relates to a composition for the prophylaxis and treatment of muscle-related diseases containing fresh vinegar extract or a compound isolated therefrom as an active ingredient. (1) Significantly increased MyoD transcriptional activity and differentiated it through (1) MyoD transcriptional activity evaluation method (Experimental Example 1), which measures myoD transcriptional activity of the fresh vinegar extract of the present invention. It was found to promote MHC expression in C2C12 cells; (2) Effect on myoblast differentiation The number of cylinder-shaped multinucleated myotubes was increased by sample processing through experiments (Experimental Example 2), and multinucleated myotubes ( Increasing the ratio of multinuclear myotubes, it has been proved to promote myoblast differentiation to form myotubes, the results of the above experiments showed that the extract of the present invention is atony, muscle atrophy, muscular dystrophy The composition can be used as a therapeutic agent or adjuvant for muscle diseases such as muscle dystrophy, muscle degeneration, myopathy, atrophic atrophy, ataxia, cachexia and sarcopenia. It can be usefully used as a therapeutic pharmaceutical composition, health functional food and health supplement food.

Description

A composition comprising an extract of Angelica keiskei for treating and preventing muscle-related disorder}

The present invention relates to a composition for the prevention and treatment of muscle-related diseases containing fresh vinegar 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

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

Document 5 J Biol. Chem 278: 40717-40722, 2003

Document 6 Cell Mol Life Sci 70: 4117-4130, 2013

Sci Signal 6: re2, 2013

8 Langley, B .; Thomas, M .; Bishop, A .; Sharma, M .; Gilmour, S .; Kambadur, R. J. Biol. Chem. 2002, 277, 49831

Document 9 Biol. Chem 2004, 279 , pp.52643

10 Eur J Histochem. 2004, 48, 223-33

11 Chem. Biol. Interact. 2016, 248 , 60

12. Histochem. Cell Biol. 2012 , 138, p. 187;

13 J. Cell Physiol. 2010 , 224, p7

14 J. Biol. Chem 2004, 279 , 52643

Document 15 Chem. Biol. Interact. 2016, 248 , 60.

16 Sci Rep. 2016, 11, 24214.doi: 10.1038 / srep24214

The present invention relates to a composition for the prevention and treatment of muscle-related diseases containing fresh vinegar extract or a compound isolated therefrom.

Muscle regeneration includes atony, muscular atrophy, muscular dystrophy, muscle degeneration, myopathy, muscular atrophy, ataxia, cachexia and sarcopenia It has been noted for the purpose of overcoming skeletal muscle degenerative diseases such as. ( J Cachexia Sarcopenia Muscle. 2015, 6, 197)

Progressive weakness and loss of function threaten the quality of life and reduce the survival of cancer patients. More than 30% of cancer patients die from weight loss due to muscle loss. This loss of muscle function is due to myo-proteins degeneration and decreased muscle fiber cross-sectional area, muscle strength, muscle number of myofibers and insulin responsiveness. It is accompanied by common.

In addition to cancer, muscle loss can also be caused by the progression of aging and various chronic diseases. As aging progresses, some of the newly created skeletal muscles are replaced with fibrous tissues, resulting in sarcopenia, which reduces the body's skeletal muscle mass and strength. In addition, muscle loss also occurs in chronic diseases in which the incidence increases with age, such as hypertension, impaired glucose tolerance, and diabetes, obesity, dyslipidemia, atherosclerosis, and cardiovascular disease. ( Pharmacol Res. 2015 , 99 , 86).

Strategies for the treatment of muscle degeneration include the inhibition of inflammatory molecules and myostatin or the activation of cyclic AMP, proliferator-activated receptor gamma coactivator (PGC) -1α and the insulin signaling system. Although a number of potential drugs have been developed, megestrol acetate is the only drug approved by the US Food and Drug Administration. Drugs for the treatment and prevention of myopathy exhibit activity that inhibits muscle protein catabolism or enhances satellite cell functions. ( Pharmacol. Res. 2015, 99, 86100).

In muscle, assimilation and catabolism are balanced and regulate muscle production. In this context, many biological signaling processes are regulated in muscle cells. When the signaling response that induces synthesis is activated rather than the degradation of muscle proteins, the synthesis of muscle proteins is increased, leading to increased muscle size (hypertrophy) or increased muscle fiber (hyperplasia) resulting in excessive muscle production (The Korea Journal of Sports Science 2011, 30, 1551).

Muscle induction factors induce muscle protein synthesis by activating the PI3K (phosphatidylinositol-3 kinase) / Akt pathway in muscle cells and thus phosphorylating downstream proteins. The activity of mTOR (mammalian target of rapamycin) by PI3K / Akt signaling is recognized as a central growth signaling mechanism that integrates various growth signals in cells. Activation of mTOR activates two subtargets, 4EBP1 (4E-binding protein) and p70S6K (phosphorylated 70-kDa ribosomal S6 kinase), which induce muscle protein synthesis to increase muscle mass (The Korea Journal of Sports Science 30: 1551-1561, 2011, J Biol. Chem 278: 40717-40722, 2003).

In addition to mTOR, differentiation and muscle formation of myocytes is regulated by a variety of factors (cell Mol Life Sci 70: 4117-4130, 2013). Among them, myoD initiates the expression of specific genes related to muscle differentiation, inducing mesenchymal stem cells to differentiate into myoblasts. Myogenin and myosin heavy chain (MHC), which are controlled by MyoD, induce fusion of myoblasts, leading to the formation of myotubes and muscle fibers. Muscle fibers formed through this process are bundled to finally form muscles (Cell Mol Life Sci 70: 4117-4130, 2013; Sci Signal 6: re2, 2013).

Under normal conditions, the quiescent satellite cells as primary stem cells continue to proliferate and differentiate. Injured muscle secretes a variety of growth factors that activate the proliferation of myogenic satellite cells, called myocytes. Activated myoblasts 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 myogenic lineage and play an important role in initiating myoblast 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-muscular specific factors such as E protein, Mef-2 family proteins and transcriptional cofactors.

Angelica keiskei is a dicotyledonous plant, 4-hydroxyderricin, xanthoangelol, xanthoangelol E, xanthoangelol D, and xanthoangelol D. Ingredients such as xanthoangelol F, xanthoangelol B, xanthokeismin A, and isobavachalcone are known, and are known to be effective in treating diseases such as colds, asthma and diabetes. It is called the Japanese ashitaba, the Japanese name Ashitaba, and includes Angelica keiskei , Angelica kiusina , and the like.

However, none of the literature discloses or teaches the therapeutic effect of muscle-related diseases containing fresh vinegar extract as an active ingredient.

In this regard, the present inventors have conducted MyoD transcriptional activity through a myogenic cell differentiation efficacy evaluation method (Experimental Example 1) to measure myoD transcriptional activity in fresh vinegar extract as part of a study to develop an effective prophylactic and therapeutic agent for muscle-related diseases. Whether significantly increased, or increased MHC expression in differentiated C2C12 cells by Western blotting analysis, and (2) the sample through the experiment to induce myoblast differentiation (Experimental Example 2) Treatment induced concentration-dependent differentiation into cylinder-shaped multinucleated myotubes. In addition, the number of multinuclear myotubes was increased. The results of the above experiments showed that the fresh vinegar extract of the present invention and the compound 4-hydroxyderricin, xanthoangelol, xanthoangelol, xanthoangelol D, xanthoangelol F, xanthoangelol B, xanthokeismin A, isobavachalcone, tension dystrophy, muscular dystrophy, muscular dystrophy, muscular dystrophy The present invention was completed by confirming that it can be used as a therapeutic agent or adjuvant for muscle diseases such as hypertrophy, muscular dystrophy, ataxia, myasthenia, cachexia and senile muscular dystrophy.

An object of the present invention to provide a pharmaceutical composition for the prevention and treatment of muscle-related diseases containing fresh vinegar extract or a compound isolated therefrom as an active ingredient.

In another aspect, the present invention provides a functional food for the prevention and improvement of muscle-related diseases containing fresh vinegar extract or a compound isolated therefrom as an active ingredient.

Fresh vines as defined herein are characterized by comprising roots, stems, outpost leaves, preferably roots or outposts of the same plant, such as Angelica keiskei , Angelica kiusina , and the like.

Fresh vinegar extract as defined herein is characterized in that it comprises a fresh vinegar crude extract, a polar solvent soluble extract or a non-polar solvent soluble extract purified from the crude vinegar extract.

The crude extract as defined herein is a solvent selected from water containing purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, or a mixed solvent thereof, preferably water or a mixed solvent of water and ethanol, more preferably Is characterized in that the extract available in ethanol.

Non-polar solvent soluble extraction fractions as defined herein include those extraction fractions available in non-polar solvents that refine only the extracts available in hexane, methylene chloride, chloroform, or ethyl acetate solvents from the crude extracts herein.

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

Compounds isolated from fresh vinegar extract as defined herein are isobavachalcone (compound 1), 4-hydroxyderiicin (compound 2), xanthoangelol E (compound 3), xantho Angelol D (xanthoangelol D, Compound 4), xanthoangelol (Compound 5), xanthoangelol F, (xanthoangelol F, Compound 6), xanthokesamine A (Xanthokeismin A, Compound 7), 1 -[2,4-dihydroxy-3- (6,7-dihydroxy-3,7-dimethyl-2-octenyl) phenyl] 3- (4-hydroxyphenyl-2-prefen-1- Onconcon (1- [2,4-dihydroxy-3- (6,7-dihydroxy-3,7-dimethyl-2-octenyl) phenyl] 3- (4-hydroxyphenyl-2-prepen-1-onechalcone compound 8) , 1- [2-hydroxy-3- (7-hydroxy-3,7-dimethyll-2, 5-octadienyl) -4-methoxyphenyl] -3- (4-hydroxyphenyl)- 2-propen-1-one checon (1- [2-hydroxy-3- (7-hydroxy-3,7-dimethyl-2, 5-ocadienyl) -4-methoxyphenyl] -3- (4-hydroxyphenyl)- 2-propen-1-one chalcone, compound 9), xanthoangelol B (Xant hoangolol B, compound 10).

High performance liquid chromatography (HPLC) analysis of fresh vinegar extracts and compounds as defined herein is performed under conditions of mixing an acidic solvent with a nonpolar solvent such as methanol or acetenitrile.

Muscle-related disorders as defined herein include atony, muscular atrophy, muscular dystrophy, muscle degeneration, myopathy, muscular atrophy, ataxia, cachexia and sarcopenia At least one muscle disease selected from the group consisting of muscle specific diseases caused by senile muscular atrophy or cancer, more specifically, muscular atrophy due to senile muscular atrophy or cancer, muscular dystrophy, Muscle degeneration, myopathy, muscular dystrophy, ataxia, ataxia, cachexia, sarcopenia and muscular loss.

The present invention also provides a prophylactic and anticancer adjuvant therapy or a therapeutic agent for muscle-related diseases caused by cancer containing fresh vinegar extract or a compound isolated therefrom.

The present invention also provides a pharmaceutical composition for preventing and treating muscle-related diseases caused by cancer, comprising a combination of Sinchocho extract or a compound separated therefrom and an existing anticancer agent, an anticancer adjuvant or a health functional food.

Existing anticancer agents as defined herein include cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, mustine, vicristine, procarba Procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, cisplatin, epirubicin, cispool Cisplatin, capecitabine, oxaliplatin and the like.

Cancer diseases as defined herein include leukemia, lymphoma, myeloma, myelodysplastic syndrome, breast cancer, head and neck cancer, esophageal cancer, gastric cancer, colon cancer (= colon cancer), rectal cancer, anal cancer, hepatocellular cancer, bile duct cancer, gallbladder cancer, pancreatic cancer, lung cancer (non-small cell Sex 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), primary site unknown cancer, mesothelioma, melanoma, Neuroendocrine tumors, skin cancers, hematological cancers, and the like.

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

Extracts of the present invention can be obtained by the preparation method as follows.

For example, the present invention will be described in detail below.

Fresh vinegar extract of the present invention can be prepared as follows. After washing and rinsing the dried fresh vinegar, a solvent selected from water including purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol and butanol, or a mixed solvent thereof, preferably a mixed solvent of water and ethanol, more preferably 30 ~ 100% ethanol several times and then ultrasonic extraction, hydrothermal extraction, room temperature extraction for 30 minutes to 48 hours, preferably 24 to 36 hours at a temperature of 30 ℃ to 150 ℃, preferably 50 ℃ to 100 ℃ or The crude extract of the present invention can be obtained by filtering, concentrating under reduced pressure, and drying the extract obtained by reflux extraction, preferably at room temperature extraction, about 1 to 20 times, preferably 2 to 10 times.

In addition, the polar or non-polar solvent soluble extract of the present invention is added to the water of about 0.0005 to 500 times, preferably 0.05 to 5 times the volume (v / w%) of the crude extract obtained above, n-hexane, Non-polar solvent soluble extract fractions which are available in non-polar solvents such as n-hexane, methylene chloride, ethyl acetate by performing a conventional fractionation process using methylene chloride, ethyl acetate and butanol; And polar solvent soluble extraction fractions soluble in polar solvents such as butanol and water, respectively.

The compounds of the present invention can be prepared as follows. For example, the crude fresh extract obtained above is fractionated with ethyl ether. Silica gel open column chromatography, flash column chromatography, RP C18 column using the method of increasing the polarity by starting the distillation of ethyl acetate soluble fraction with n-hexane to mobile phase and increasing the polarity to acetone (Acetone). Purification using chromatography or chromatography such as Diaion HP-20 column chromatography may be performed several times selectively to purify and obtain the compounds of the present invention, respectively.

The present inventors pay attention to MyoD transcriptional activity (Experimental Example 1) through the differentiation promoting efficacy evaluation method (Experimental Example 1) of measuring the myoD transcriptional activity of the extract obtained by the above production method (Experimental Example 1). It was confirmed that MHC expression was increased in the C2C12 cells, and (2) the effect of myoblast differentiation on the myoblast differentiation (Experimental Example 2). shaped) increased the number of multinucleated myotubes, and proved to promote myotube formation through myoblast differentiation, confirming that the composition is useful as a pharmaceutical composition or health functional food for the prevention and treatment of muscle diseases.

Accordingly, the present invention provides a pharmaceutical composition or health functional food for preventing and treating muscle-related diseases containing the fresh vinegar extract obtained by the above method or a compound isolated therefrom as an active ingredient.

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

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

In this case, organic acids and inorganic acids may be used as the free acid, and hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, and the like may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, and trifluoroacetic acid may be used as the organic acid. , 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 (glutaric acid), glucuronic acid (glucuronic acid), aspartic acid, ascorbic acid, carbonic acid, vanic acid and hydroiodic acid may be used.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the invention include salts of acidic or basic groups which may be present in the compounds of the invention, unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts. It can be prepared through.

The composition of the present invention comprises 0.01 to 99% by weight of the extract or compound relative to the total weight of the composition.

However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

Compositions comprising extracts or compounds of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Compositions comprising extracts or compounds according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods Carriers, excipients and diluents that may be included in the formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one or more excipients in at least one of the excipients, cotton, starch, calcium carbonate, sucrose ( It is prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts or compounds of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract or compound is preferably administered at 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. Administration may be administered once a day or may be divided several times. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

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

The present invention also provides a therapeutic agent for senile muscular dystrophy or cachexia containing the fresh vinegar extract or a compound isolated therefrom as an active ingredient, and the therapeutic agent is for improving the effect of weight change or appetite recovery in combination therapy with senile muscular atrophy or cachexia. In addition, the present invention provides a combination therapy using the composition of the present invention rather than an anticancer agent alone in cachexia treatment.

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

As defined herein, "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" means It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on structure and function.

The dietary supplement for the prevention and improvement of the muscle-related diseases of the present invention comprises the extract or compound in an amount of 0.01 to 95%, preferably 1 to 80% by weight, based on the total weight of the composition.

Furthermore, the composition of the present invention may be a dietary supplement or dietary supplement for the purpose of treating and improving muscle-related diseases.

In addition, as a health functional food in the form of a pharmaceutical dosage form such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups or tea bags, leaching tea, health drinks for the purpose of preventing and improving muscle-related diseases. Manufacturing and processing are possible.

The present invention also provides a dietary supplement for the prevention and improvement of muscle-related diseases containing fresh vinegar extract or a compound isolated therefrom as an active ingredient.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extract as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. have. The proportion of the natural carbohydrate is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

The present invention also provides a food or food additive for the prevention and improvement of muscle-related diseases containing fresh vinegar extract or a compound isolated therefrom as an active ingredient.

When the extract or compound according to the present invention is used as a food additive, the extract may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, etc., includes all of the health food in the conventional sense.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the extract or compound of the present invention may be added to food or beverage for the purpose of preventing the disease of interest. At this time, the amount of the extract or compound in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be added.

(1) Significantly increased MyoD transcriptional activity and differentiated C2C12 cells through (1) MyoD transcriptional activity evaluation method (Experimental Example 1) of myoD extracts and compounds of the present invention to measure myoD transcriptional activity. It was confirmed that the induction of MHC expression in promotes the differentiation into muscle cells; (2) Influence on myoblast differentiation (Experimental Example 2) Induced differentiation into cylinder-shaped multinucleated myotubes in a concentration-dependent manner by multiplying sample through multi-nucleus. It was confirmed that the number of multinucleated myotubes increased, these experimental results indicate that the samples of the present invention are atony, muscular atrophy, muscular dystrophy, muscle degeneration, myopathy , By confirming that the composition can be used as a therapeutic agent or adjuvant for muscle diseases such as muscular dystrophy, ataxia, myasthenia, cachexia and sarcopenia, the composition for the prevention and treatment of muscle diseases, health functional foods and It was found to be useful as a health supplement.

1 is a diagram showing the structure of a compound isolated from the extract of the present invention.
2 is a diagram showing an HPLC chromatogram for determining the content of compound 2 and compound 5 contained in the extract of the present invention.
Figure 3 is a diagram showing myoD transactivation of the extract of the present invention. (Data is expressed as mean ± SD, meaning * p <0.001).
Figure 4 is a diagram showing the effect on the protein expression of MHC (myosin heavy chain) of the extract of the present invention (data denoted by mean ± SD means * p <0.05).
5 is a diagram showing the effect on the expression of MHC (myosin heavy chain) in multinucleated myotubes.
Figure 6 is a diagram showing the effect on the myoD transactivation and MHC (myosin heavy chain) expression of the compounds contained in the extract of the present invention (data denotes mean ± SD * p <0.001 means).
Figure 7 is a diagram showing the effect on the protein expression of MHC (myosin heavy chain) of the compounds contained in the extract of the present invention. (Data is expressed as mean ± SD, meaning * p <0.05.)
8 is a diagram showing the effect on the protein expression of MHC (myosin heavy chain) of chalcone (chalcone) compound 1, compound 2, compound 5, compound 6 of the compounds included in the extract of the present invention (data is mean ± SD * P <0.05).
9 is a diagram showing the effect on the expression of MHC (myosin heavy chain) in multinucleated myotubes.

Hereinafter, the present invention will be described in detail by the following Reference Examples and Experimental Examples.

However, the following Reference Examples and Experimental Examples are merely illustrative of the present invention, the content of the present invention is not limited by the following Reference Examples and Experimental Examples.

Example 1. Preparation of fresh vinegar crude extract

6 liters of 100% ethanol was added to 3 kg of dried fresh vinegar (Saengdong Nongsan, Korea, Seoul, Korea), and 100% ethanol was extracted for 24 hours, filtered and concentrated under reduced pressure. The concentrated ethanol extract was lyophilized with a freeze dryer (ScanVac, Labogene) under reduced pressure to obtain 300 g of fresh ethanol extract powder (hereinafter referred to as AKCE) and used for the next experiment.

Example 2 Preparation Example of Fresh Herb Fraction

To 300 g of dried fresh ethanol extract obtained in Example 1, 500 ml of purified water was added and suspended. Were added sequentially and fractionated and dried under reduced pressure, respectively, to obtain n-hexane soluble fraction (hereinafter referred to as AKH), ethyl acetate soluble fraction (hereinafter referred to as AKE) and butanol soluble fraction (hereinafter referred to as AKB) to 14 g and 7.8 g. , 30g were each obtained and lyophilized.

Example 3. Isolation of Active Compounds from Fresh Creeper Extract

To identify the active substances, silica gel column chromatography was performed on n-Hexane / acetone gradient system (50: 1) for 21.8 g of n-hexane and ethyl acetate soluble fraction in the most active fractions. → 1: 2) to obtain nine subfractions,

Silica gel column chromatography was performed on the double fraction (F3) under Hexane / ethylacetate gradient system (10: 1 → 1: 1) solvent conditions, and F3-2 of the fraction was further analyzed. Reversed phase chromatography chromatography MeOH gradient (50% → 100%) was carried out under the solvent conditions. Among them, fractions (F3-2-5) were further subjected to silica gel column chromatography under Hexane / ethylacetate gradient (20: 1 → 1: 1) solvent conditions, and fractions F3-2-5. Silica gel column chromatography on -3 was performed by Hexane / (10% isopropanol) ethylacetate under 7: 1 solvent conditions to obtain the following compound 2 (4-hydroxyderricin, 35 mg).

Silica gel column chromatography on double fraction (F5) was further performed with Hexane / (10% isopropanol) ethylacetate under 7: 1 solvent conditions, and fraction F5-2 was subjected to reverse phase column chromatography with MeOH gradient. (gradient system, 60% → 100%) was carried out under solvent conditions to give the following compound 5 (xanthoangelol, 25 mg).

(A) Compound 2: 4-hydroxyderricin

Appearance: Yellow Powder

1 H-NMR (400 MHz, CDCl 3): δ 1.69 (3H, s, H-5 ”), 1.81 (3H, s, H-4”), 3.40 (2H, D, J = 6.8 Hz, H-1 ”) , 3.92 (3H, s, OMe), 5.24 (1H, tq, J = 7.2, 1.6 Hz, H-2 ”), 6.51 (1H, d, J = 8,8 Hz, H-5 '), 6.89 (2H , d, J = 8.8Hz, H-3, H-5), 7.47 (1H, d, J = 15.6Hz, H-α), 7.56 (2H, J = 8.8Hz, H-2, H-6) , 7.80 (1H, d, J = 8.8 Hz, H-6 '), 7.83 (1H, d, J = 15.6 Hz, H-β)

13C-NMR (100MHz, CDCl3): δ 17.8 (C-4 ”), 21.7 (C-1”), 25.8 (C-5 ”), 55.8 (Ome), 102.1 (C-5 '), 114.6 (C -1 '), 116.0 (C-3, C-5), 117.6 (C-3'), 118.1 (C-α), 122.0 (C-2 ”), 127.7 (C-1), 129.2 (C- 6 '), 130.5 (C-2, C-6), 131.9 (C-3 ”), 144.1 (C-β), 158.1 (C-4), 163.0 (C-2'), 163.3 (C-4 '), 192.4 (C = 0)

(B) Compound 5: Xanthoangelol

Appearance: Yellow Powder

1 H-NMR (400 MHz, CDCl 3): 1.50 (3H, s, H-10 ”), 1.57 (3H, s, H-9”), 1.73 (3H, s, H-8 ”), 1.59 (2H, m , H-4 ”), 2.00 (2H, m, H-5”), 3.36 (2H, d, J = 6.8 Hz, H-1 ”), 5.00 (1H, m, H-6”), 5.22 ( 1H, m, H-2 ”), 6.36 (1H, d J = 8.8 Hz, H-5 '), 6.79 (2H, d, J = 8.4 Hz, H-3, H-5), 7.36 (1H, d, J = 15.2 Hz, H-α, 7.45 (2H, d, J = 8.4 Hz, H-2, H-6), 7.60 (1H, d, J = 8.8 Hz, H-6 '), 7.70 (1H, d, J = 15.2 Hz, H-β)

13C-NMR (100MHz, Cd3OD): δ 15.9 (C-10 ”), 17.38 (C-9”), 21.46 (C-8 ”), 26.5 (C-5”), 39.6 (C-4 ”), 107.2 (C-5 '), 113.4 (C-1'), 115.3 (C-3 '), 115.8 (C-3, C-5), 117.4 (C-α), 121.7 (C-2 ”), 124.2 (C-6 ”), 126.5 (C-1), 128.8 (C-6 '), 130.3 (C-2, C-6), 131.2 (C-7”), 135.9 (C-3 ”), 144.0 (C-β), 159.4 (C-4), 161.8 (C-2 '), 163.6 (C-4'), 192.1 (C = 0)

Experimental Example 1. Component Analysis of Sinchocho extract

HPLC analysis was performed to determine the contents of Compound 2 and Compound 5 of the fresh ethanol extract of the Example.

The fresh ethanol extract of Example 1 and the compounds 2 and 5 obtained in Example 3 were prepared using a column (C18 4.6 mm x 150 mm, 5 μm, cat. 5020-01124, GL sciences Inc.) at 80% methanol. Analysis at a wavelength of 360 nm. Through this analysis, the content of Compound 2 and Compound 5 contained in the extract was analyzed.

1-1. Experiment process

HPLC was measured at 360 nm using a 4.6 x 150 mm, 5um column at 80% methanol. Samples of fresh ethanol extract and compounds 2 and 5 were prepared at 1 mg / ml, 0.1 mg / ml, and 0.01 mg / ml, respectively, and 5 µl, 2 µl, and 3 µl were injected to obtain chromatograms. The contents of the included compounds 2 and 5 were obtained.

1-2. Experiment result

As a result, the compound 2 and compound 5 contained in 5 μg of fresh vinegar extract were identified as 0.195 μg and 0.295 μg, respectively, and when the contents were obtained, the compounds 2 and 5 were identified as 3.8% and 5.8%, respectively. (Figure 2)

Experimental Example 1.Evaluation of Differentiation Promoting Effect of Myofibrillar Radix Cells

As described in the literature, the reporter vector (MyoD-responsive) in which myoD-reactive gene and luciferase were fused to C2C12 cells, a mouse myoblast cell line, to investigate the effect on the myogenic effect of ethanol extract of fresh ethanol extract of the above example. reporter 4RTK-luc) was inserted and myoD transcriptional activity was analyzed by measuring luciferase activity change according to extract treatment to evaluate the differentiation-promoting efficacy of myoblasts ( J. Biol. Chem 2004, 279 , pp. 52643).

1-1. Experiment process

A self-made reporter vector (MyoD-responsive reporter 4RTK-luciferase construct) comprising four E-box sites fused to a thymidine kinase promoter was used to assess MyoD transcriptional activity.

A myoD expression vector and a reporter vector (MyoD-responsive reporter 4RTK-luc) (provided by Prof. Kyu-Woon Bae, College of Pharmacy, Sookmyung Women's University) Transfection was carried out by the method described in ( Eur J Histochem. 2004, 48, 223-33).

After 24 hours of transfection, the cells were treated with fresh vinegar extract (10, 100, 1000 ng / ml), and further cultured for 24 hours.

The cultured cells were washed twice with phosphate buffered saline solution, lysed with reporter lysis buffer (Cat #. E1941, Promega), and then 5 μl of the cell lysate was taken into a 96-well plate for luciferase analysis (Cat # 31196, SPL) and absorbance was measured by adding 30 μl of Luciferase Substrate (Dual Luciferase Reporter Assay System, Cat # E1910, Promega).

β-galactosidase activity was measured for absorbance by adding 30 μl of Stop and Glo reagent (Ccat # E1910, Promega) to the cell lysate. Data are expressed as relative luciferase activity (RLU) divided by β-galactosidase activity and expressed as mean ± SD.

1-2. Experiment result

As a result of the experiment, when luciferase activity in control cells was set to 1, 3 times of fresh vinegar extract was treated with 10 ng / ml, 4 times with 100 ng / ml, and 13 times with 1000 ng / ml. In addition, it was confirmed that the fresh vinegar extract increased MyoD transcriptional activity (Table 1 and Figure 3) in a concentration-dependent manner.

Luciferase activity test results of the extracts (FIG. 3) Fresh vinegar extract concentration (ng / ml) MyoD Reporter Activity (pear) Control 1.0 10 3.0 100 3.7 1000 12.8

Experimental Example 2. Effect of fresh vinegar extract on myoblast differentiation

In order to determine the effect on myoblast differentiation of fresh vinegar extract of the above example, the experiment was performed using the evaluation method described in the literature as described below ( Chem. Biol. Interact. 2016, 248 , 60).

In the process of differentiation of myoblasts into myotubes, mononucleated myoblasts are elongated and transformed into cylinder-shaped myocytes. In activated myocytes, the expression of MyoD, myogenic factor (Myf) -5, myogenin, and myogenic regulatory factor (Mrf) -4 is increased to activate genes involved in different muscle differentiation and promote differentiation into myotube fibers. . The resulting myocytes fuse with each other to form multinucleated myotubes, which increase the expression of Mrf-4, myogenin, or MHC, a muscle differentiation marker ( Histochem. Cell Biol. 2012 , 138, p. 187; J. Cell Physiol. 2010 , 224, p7).

Therefore, MHC is known as a marker of late stage of muscle differentiation, and the level of myogenic differentiation ability of myoblasts can be predicted by analyzing the level of MHC expression in terminally differentiated myotubes.

Therefore, in this experiment, MHC protein expression was measured and used as an indicator of the differentiation of myoblasts into myotubes.

2-1. Experiment process

C2C12 myoblasts were treated with fresh vinegar extract (10, 100, 1000 ng / ml) to induce differentiation by treatment on differentiation medium (2% horse serum-containing DMEM, Cat # 11965-084, Gibco) for 3 days. .

Cell lysates were obtained from differentiated cells to evaluate the expression level of MHC protein by Western blot (FIG. 4A), mouse anti-MHC, Santa cruze and anti-mouse IgG2b Alexa- Immunostaining was performed with fluor 568) to evaluate MHC expression (FIG. 2B).

For Western blot analysis, approximately 3 X 10 ⁴ myoblasts were incubated in a 60 mm plate for 24 hours, and then, each sample was added to differentiation medium for differentiation for 3 days, followed by 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 (Protein extracts), 25 μg of protein extracts were subjected to SDS-polyacrylamide gel electorphoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes, to which the primary mouse anti-MHC was bound for 12 hours at 4 ° C. After binding to horseradish peroxidase-linked anti mouse secondary antibody (goat anti-mouse IgG-HRP, Enzo), the amount of MHC protein was analyzed by chemiluminescence, and pan-cadherin (Cat) was used as a loading control. # 3678, Sigma).

In the same manner as described above, the source cells were differentiated in the differentiation medium to which each sample was added, and immunofluorescence staining was performed. Each differentiation medium was removed, washed twice with phosphate buffered saline, and fixed for 20 minutes with 4% paraformaldehyde. Again washed with phosphate buffered saline twice, and treated with 0.1% tritonX-100 for 20 minutes. After washing twice with phosphate buffered saline, blocking in 5% horse serum solution, 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 then MHC expression was analyzed using Alexa Flouor 568-bound secondary anti-mouse (A-21144, MicoProbes) and DAPI (D9542, Sigma).

Immunfluorescence results of MHC-positive myotubes were visualized in red and DAPI-labeled nuclei were visualized in blue.

Multinucleated ( > 5) MHC-expressing myotubes were generated at randomly selected sites (Scale bar = 200 mm). After analyzing MHC-positive and multinucleated myotubes, total cells were used to calculate the relative multinucleated-MHC-positive myotubes in each cell. .

2-2. Experimental Results (Tables 2 to 3)

In FIG. 4A, the effect on MHC expression of fresh vinegar extract was measured. The differentiation medium of myoblasts was treated at concentrations of 10, 100, 1000 ng / ml and differentiated into root canal fibers for 3 days, and the cells were harvested. The protein expression of MHC, a marker for differentiation into root canal fibers, was analyzed by Western blotting analysis. .

As a result, when MHC expression was 1 in the control cells, the fresh vinegar extract was increased three times when treated with 10 ng / ml, four times when treated with 100 ng / ml, and five times when treated with 1000 ng / ml.

In accordance with the results of Myo D transcriptional activation experiments, the extract of Sinchochoa was found to increase the expression of MHC as well as myoD in differentiated C2C12 cells.

These experimental results confirmed that the fresh vinegar extract of the present invention promotes myogenic differentiation in a concentration-dependent manner.

In Figure 5, the muscle differentiation activity of the extract was measured by immunofluorescence staining with anti-MHC antibodies (antibodies) and DAPI.

As shown in FIG. 5, increased red-fluorescence means that the fresh herb extract promotes MHC expression in C2C12 cells in a concentration-dependent manner, and the cylindrical form of MHC expressed through DAPI counter staining. (cylinder-shaped) root canal cells were confirmed to be multinucleated (multinucleated).

Myoblast differentiation in this experiment was expressed as a percentage of multinucleated MHC-positive cells. As a result, it was found that the extract of Sinchochoa increased MHC expression and the number of cylinder-shaped multinucleated myotubes in the root canal cells even at a low concentration of 1000 ng / ml and activated muscle cell differentiation. Proved.

MHC expression test results of the extracts (FIG. 4) Fresh vinegar extract concentration (ng / ml) MHC / Pan-cad (ship) Control 1.0 10 3.2 100 3.7 1000 4.5

Result of muscle differentiation activity of the extract (FIG. 5) Fresh vinegar extract concentration (ng / ml) Multinuclear cell count (fold) Control 1.0 10 16.1 100 28.2 1000 32.1

Experimental Example 3. Evaluation of Differentiation Promoting Effect of Myofibrillar Cells

In order to examine the effect on the myogenic effect of myoblasts of the compounds contained in fresh vinegar of the above embodiment, as described in the literature, a reporter vector fused with myoD-reactive gene and luciferase to C2C12 cells, a mouse myoblast cell line ( MyoD-responsive reporter 4RTK-luc) was inserted and myoD transcriptional activity was analyzed by measuring luciferase activity change according to compound treatment to evaluate the differentiation-promoting effect of myoblasts ( J. Biol. Chem 2004, 279 , 52643).

3-1. Experiment process

The experiment was carried out in the same manner as in Experiment 1-1.

3-2. Experiment result

As a result of the experiment, when luciferase activity in the control cell was set to 1, it was confirmed that the compound 2 and the compound 5 exhibited 21-fold and 13-fold activities when 0.1 nm of the fresh herb compound was treated, respectively. Other compounds were also found to significantly increase MyoD transcriptional activity (Table 4 and FIG. 6).

Experimental results of MyoD transcriptional activity of compounds (FIG. 6) Treatment group MyoD Reporter Activity (pear) Control 1.0 Compound 1 12.8 Compound 2 22.1 Compound 3 6.0 Compound 4 2.0 Compound 5 14.1 Compound 6 7.9 Compound 7 10.0 Compound 8 13.1 Compound 9 6.1 Compound 10 14.2

Experimental Example 4.  Effect of Fresh Creeping Compounds on Myoblast Differentiation

In order to determine the effect on myoblast differentiation of the compound contained in fresh vinegar of the above example, the experiment was carried out as follows using the evaluation method described in the literature ( Chem. Biol. Interact. 2016 , 248 , 60 .).

In this experiment, MHC protein expression was measured and used as an indicator for the differentiation of myoblasts into myotubes.

4-1. Experiment process

The same procedure as in Experiment 2-1 was conducted.

4-2. Experimental Results (Table 5)

Figure 6 shows the effect on the MHC expression of the compounds contained in fresh vinegar. Cells were harvested by differentiation of myoblasts in differentiation medium at a concentration of 0.1 nm for 3 days, and cells were harvested. Protein expression of MHC, a marker of differentiation into myotubes, was analyzed by Western blotting.

As a result, when MHC expression was 1 in the control cells, when the compound 2 and compound 5 were treated, the expressed MHC was increased 6-fold and 3-fold, respectively.

Compounds contained in fresh vinegar were found to increase the expression of MHC as well as myoD in differentiated C2C12 cells, consistent with Myo D transcriptional activation experiments.

These experimental results confirmed that the compound contained in the fresh vinegar of the present invention promotes myogenic differentiation (myogenic differentiation).

Results of the effects on the expression of MHC compounds (Fig. 7) Treatment group MHC / Pan-cad (ship) Control 1.0 Compound 1 2.9 Compound 2 5.7 Compound 3 2.0 Compound 4 1.1 Compound 5 2.7 Compound 6 1.7 Compound 7 2.0 Compound 8 1.0 Compound 9 1.4 Compound 10 0.7

Experimental Example 5. Effect on myoblast differentiation of chalcone compounds

In order to determine the effect on the myoblast differentiation of several kinds of chalcone compounds contained in fresh vinegar of the above example, the experiment was carried out as follows using the evaluation method described in the literature ( Chem. Biol. Interact. 2016) . , 248 , 60.).

In this experiment, MHC protein expression was measured and used as an indicator for the differentiation of myoblasts into myotubes.

4-1. Experiment process

The same procedure as in Experiment 2-1 was conducted.

4-2. Experimental Results (Tables 6-7)

8 and 9 show the effect on the MHC expression of several kinds of chalcone compounds contained in fresh vinegar. Cells were harvested by differentiation of myoblasts in differentiation medium at a concentration of 0.1 nm for 3 days, and cells were harvested. Protein expression of MHC, a marker of differentiation into myotubes, was analyzed by Western blotting.

As a result, when MHC expression in control cells was set to 1, MHC expressed by treatment with a chalcone compound increased by 3-5 times.

As shown in FIG. 9, increased red-fluorescence means that the chalcone compounds contained in fresh vines promote MHC expression in C2C12 cells, and MHC through DAPI counter staining. It was confirmed that the expressed cylindrical cylinder-shaped root canal cells are multinucleated.

Results of the effect on the expression of MHC compounds (Fig. 8) Treatment group MHC / Pan-cad (ship) Control 1.0 Compound 1 1.6 Compound 2 4.2 Compound 5 3.3 Compound 6 2.8

Muscle differentiation activity test results of the extract (Fig. 9) Treatment group Multinuclear cell count (fold) Control 1.0 Compound 1 1.7 Compound 2 2.5 Compound 5 2.0 Compound 6 1.5

Hereinafter, the preparation examples of the composition including the sample extract of the present invention, but the present invention is not intended to limit it, only intended to explain in detail.

Formulation Example 1 Preparation of Powder

Extract (AKCE) ------------------------------------------- 20 mg

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

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

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

Fraction (AKH) -------------------------------------------- 10 mg

Corn Starch -------------------------------------------- 100 mg

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

Magnesium Stearate ------------------------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

Fraction (AKE) ------------------------------------------- 10 mg

Crystalline Cellulose -------------------------------------- 3 mg

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

Magnesium Stearate --------------------------------- 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

Fraction (AKB) -------------------------------------------- 10 mg

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

Sterile Distilled Water for Injection ----------------------------------- 2974 mg

Na ₂ HPO ₄ , 12H ₂ O ------------------------------------------ -26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Extract (AKCE) ------------------------------------------- 20 mg

Isomerized sugar ------------------------------------------------ -10 g

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

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

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, the lemon flavor is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Food

Extract (AKCE) ---------------------------------------- 1000 mg

Vitamin Mixture -------------------------------------------- Proper

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

Vitamin E ----------------------------------------------- 1.0 Mg

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

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

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

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

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

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

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

Folic Acid ------------------------------------------------- --- 50 μg

Calcium Pantothenate ------------------------------------------ 0.5 mg

Mineral mixture -------------------------------------------- Correct

Ferrous Sulfate --------------------------------------------- 1.75 Mg

Zinc Oxide ---------------------------------------------- 0.82 mg

Magnesium Carbonate ------------------------------------------ 25.3 mg

Potassium monophosphate --------------------------------------------- 15 Mg

Dicalcium Phosphate --------------------------------------------- 55 mg

Potassium Citrate ---------------------------------------------- 90 mg

Calcium Carbonate ----------------------------------------------- 100 Mg

Magnesium Chloride ------------------------------------------ 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 7 Preparation of Healthy Drink

Compound 1 ------------------------------------------------ 1000 mg

Citric Acid ------------------------------------------------- 1000 mg

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

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

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

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

After mixing the above components according to a conventional healthy beverage production method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated and then stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is a composition that is relatively suitable for the preferred beverage in a preferred embodiment, the compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

Claims (3)

After washing and rinsing the dried Angelica keiskei roots, 30-100% ethanol was mixed several times, followed by repeating room temperature extraction for 2 to 10 times at a temperature of 50 ° C to 100 ° C for 24 to 36 hours. A pharmaceutical composition for the prevention and treatment of senile muscular dystrophy or cachexia containing 30-100% ethanol extract of Angelica keiskei root obtained by filtration, concentration under reduced pressure, and drying the obtained extract. After washing and rinsing the dried Angelica keiskei roots, 30-100% ethanol was mixed several times, followed by repeating room temperature extraction for 2 to 10 times at a temperature of 50 ° C to 100 ° C for 24 to 36 hours. A health functional food for the prevention and improvement of senile muscular dystrophy or cachexia containing 30-100% ethanol extract of Angelica keiskei root obtained by filtration, concentration under reduced pressure, and drying the obtained extract. delete

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