KR102383194B1 - A pharmaceutical composition for prevention or treatment of muscle atrophy comprising the Sinomenium acutum extract or fractions thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for improving, preventing, or treating muscle atrophy or muscle loss comprising cynomenin and/or acutumin obtained from a Bangui extract or a fraction thereof as an active ingredient. The composition of the present invention has the effect of improving, preventing or treating muscle loss and muscle atrophy by increasing the expression level/activity level of the protein involved in muscle hypertrophy, and has the advantage of having no side effects by using a substance derived from a natural product.

Description

A pharmaceutical composition for the prevention or treatment of muscle atrophy comprising the Bangui extract or fractions thereof as an active ingredient {A pharmaceutical composition for prevention or treatment of muscle atrophy comprising the Sinomenium acutum extract or fractions thereof as an active ingredient}

The present invention relates to a composition for improving, preventing, or treating muscle atrophy or muscle loss comprising cynomenin and/or acutumin obtained from a Bangui extract or a fraction thereof as an active ingredient.

Bangi ( Sinomenium acutum ) is sour, living (散), painful (苦), and non-toxic. It is effective against wind and humidity, and has the effects of dehumidifying and dehumidifying. In addition, it is strong and descending due to high cold, and has the effect of dehydration and excretion. Therefore, it is used for watery and cessation of water. It is mainly applied to symptoms of rectification of water in the lower body . Because it is a great anticipation (大苦大寒) and it is easy to damage a crisis, it should be used with caution for those who are yin-heo and those who are not-risk (脾胃气虛) (Benchohak, 1995). It has been reported that Banggi Hwanggi-tang and Banggi Bokryeong-tang as an herbal preparation with Banggi showed efficacy in hyperlipidemia (Son Young-Jong & Lee Young-Jong, 2003; Ki Sung-Sik & Lee Young-Jong, 2005).

As major components, sinomenine, magnoflorine, acutumine, and syringaresinol have been reported (Zhao et al ., 2005). The pharmacological action of sinomenine is most widely known, and it has been reported that it is used in rheumatoid arthritis among autoimmune diseases (Wang et al ., 2011; Liu et al ., 1994). Prevention of galactosamine, lipopolysaccharide (LPS)-induced liver failure by inhibiting the production of tumor necrosis factor (TNF) and reactive oxygen species (Kondo et al ., 1994) and Treg cells in intestinal lymph nodes An antirheumatic arthritis effect was reported by regulating the ratio of and Th17 cells (Tong et al ., 2015). It has been reported that Magnoflorine has an anti-diabetic effect by inhibiting lipid peroxidation of high density lipoprotein (HDL) and protecting HDL (Hung et al. , 2007) and α-glucosidase (Pastel et al.) al ., 2012).

On the other hand, muscles can be divided into skeletal muscles, smooth muscles, and cardiac muscles in terms of structure and function. The skeletal muscles are about 600 voluntary muscles that are located just under the skin of the hands, feet, chest, and stomach and are attached to bones through bones or tendons of the whole body. Suitable for moving or supporting bones through contraction. Contraction occurs and is regulated by nerve signals. It accounts for 40-50% of body weight and has functions such as maintaining body temperature and generating energy. The microfibrils, actin and myosin, are arranged in a regular arrangement, and horizontal patterns can be observed under a microscope (Lieber R. L., 2002; Edwards R. H., 1981).

Skeletal muscle fibers are biochemically classified according to the content of mitochondria, and are divided into Type I, Type IIa, and Type IIb. Posture maintenance muscle, which consists of red slow muscle fibers and maintains a weak force for a long time, is called Type I. It is a muscle suitable for exercise such as aerobic long-distance running due to its high content of mitochondria. Among fast muscle fibers, those having the characteristics of slow muscle fibers are called Type IIa. When making a movement, a muscle composed of white fast-twitch fibers is used, which is called an active muscle and is classified as Type IIb. It is a muscle suitable for exercise such as anaerobic sprinting due to its low content of mitochondria. These skeletal muscle fibers are distributed in different proportions in different parts of the body (Tortora et al , 2008).

Anti-anabolic and catabolic action of unbalanced muscle fibers causes muscle atrophy. Here, muscle atrophy refers to the loss of size and mass of muscle cells and muscle tissue when the muscles are not used due to aging, disease states (excessive exposure to stress hormones, cancer, sepsis, starvation, etc.) say When muscle atrophy occurs, muscle strength for physical activity is weakened, and a vicious cycle of musculoskeletal degeneration begins. A decrease in walking speed and weakness in grip strength are the main symptoms and indicators of muscle mass loss, and can lead to falls, fractures, joint damage, metabolic disorders, and cardiovascular disease.

Glucocorticoids in our body cause molecular and biological changes in muscle fibers and are directly or indirectly involved in anti-anabolism and catabolism. As an anti-anabolic action, dexamethasone, a glucocorticoid compound, inhibits the PI3K/Akt/mTOR pathway, which inhibits the activities of downstream effectors 4E-BP1 and S6K1, such as eIF4G (Eukaryotic translation initiation). factor 4 G) and eIF4E (Eukaryotic translation initiation factor 4 E). This suppresses the mRNA translation process for protein synthesis, and it appears as a muscle fiber atrophy due to inhibition of muscle fiber synthesis and protein degradation (Shackman et al ., 2013). Dexamethasone also induces muscle atrophy by inhibiting muscle synthesis and causing protein breakdown. It expresses atrogenes (Atrogin-1, MuRF-1) that induce muscle atrophy according to the mechanism leading to 'PI3K/Akt → FOXO activation and GSK3 inactivation'. These genes are proteins represented by the ubiquitin-proteasome system. lead to decomposition.

KR 10-2004-0059243

As a result of intensive research on ingredients derived from natural products that can alleviate skeletal muscle loss, the present inventors found that Bangui extract and its fractions and Sinomenine and Acutumine obtained therefrom showed the effect of increasing the expression level of the protein related to muscle hypertrophy and C2C12 by dexamethasone. The effect of preventing a decrease in the diameter of cells was confirmed and the present invention was completed.

Accordingly, the technical problem to be achieved by the present invention is to provide a composition for improving, preventing or treating muscular atrophy comprising a banggi extract as an active ingredient.

In addition, another object of the present invention is to provide a composition for improving, preventing or treating muscular atrophy comprising at least one selected from the group consisting of sinomenine and acutumine obtained from the Bangui extract as an active ingredient will do

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention provides a pharmaceutical composition for preventing or treating muscular atrophy comprising a banggi extract as an active ingredient.

As an embodiment of the present invention, the Bangui extract is water, C ₁ to C ₄ lower alcohol, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and mixtures thereof It may be extracted with one or more solvents selected from the group consisting of solvents, preferably extracted with water and a C ₁ to C ₄ lower alcohol mixed solvent, and more preferably 75% ethanol is extracted as a solvent. it could be

As another embodiment of the present invention, when extracting the Bangui extract, an edible acid may be added to a solvent to extract, preferably acetic acid, citric acid, malic acid ( Malic acid), succinic acid (Succinic acid), fumaric acid (Fumaric acid), tartaric acid (Tartaric acid), amino acid (Amino acid), and at least one selected from the group consisting of lactic acid (Lactic acid) may be added to the solvent , more preferably acetic acid may be added.

As another embodiment of the present invention, the Bangui extract may be extracted with an ethanol solvent to which acetic acid is added, and in this case, it may be extracted with a solvent in which 0.5% acetic acid is added to 75% ethanol.

As another embodiment of the present invention, the Bangui extract may include cynomenin and/or acutumin, and when cynomenin and acutumin are included, it may be included in 2-5:1 parts by weight, respectively, preferably may be included in 3 to 4:1 parts by weight.

In addition, the present invention provides a pharmaceutical composition for preventing or improving muscular atrophy comprising at least one selected from the group consisting of sinomenine and acutumine as an active ingredient.

In one embodiment of the present invention, the composition includes cynomenin and acutumin, and may include 2 to 5 parts by weight of cynomenin based on 1 part by weight of acutumin, preferably based on 1 part by weight of acutumin As may be included in 3 to 4 parts by weight.

In addition, the present invention provides a food composition for preventing or improving muscle loss comprising a banggi extract as an active ingredient.

In addition, the present invention provides a food composition for preventing or improving muscle loss comprising cynomenin and/or acutumin as an active ingredient.

In addition, the present invention provides a method for preventing or treating muscular atrophy comprising administering a Bangui extract to an individual.

In addition, the present invention provides a method for preventing or treating muscular atrophy comprising administering cynomenin and/or acutumin to a subject.

In addition, the present invention provides the use of the Bangui extract for the preparation of a drug for the prevention or treatment of muscle atrophy.

In addition, the present invention provides the use of the Bangui extract for the preparation of a drug for the prevention or treatment of its atrophy.

The Bangui ( Sinomenium acutum ) extract and the sinomenine and acutumine components isolated therefrom having the effect of preventing and improving muscle atrophy according to the present invention increase the expression level and / or activity of proteins involved in muscle protein synthesis to prevent and improve muscle atrophy have

In particular, the Banggi ( Sinomenium acutum ) extract and sinomenine and acutumine components for the purpose of preventing and improving the symptoms of muscle atrophy are derived from natural products that have been proven to be highly safe for a long time, so there are very few or no side effects according to their use.

Therefore, the present invention can be used for the purpose of having an improvement and a preventive effect on the symptoms of muscle atrophy beyond the scope of the existing invention related to air-conditioning.

1 is a schematic diagram of a centrifugal separation chromatography system for obtaining a Bangui extract fraction.
Figure 2 is a diagram showing the results of HPLC chromatography of the Bangui extract.
3 is a photograph of observing the morphology of C2C12 myoblasts, myotube cells, and myocytes. Specifically, (A) is myoblasts after 48 hours of culture, (B) is myotube cells after 6 days of differentiation, (C) ) are myotube cells in which muscle atrophy was induced by treatment with 1 μM of Dexamethasone for 48 hours.
4 is a diagram showing the results of CPC chromatography of the Bangui extract.
5 is a diagram showing the mass of the Bangui extract fraction and the result of CPC chromatography.
6 is a schematic diagram of obtaining fractions (I to XVII) by CPC chromatography of the Bangui ethanol extract.
7 is a graph showing the results of chromatography of compounds 1 to 7 obtained by purifying fractions VI to VIII and X of the Bangui extract by prep HPLC.
8 is a structural formula of compounds 1 to 7 obtained by purifying the VI to VIII, and X fractions of the Bangui extract by prep HPLC.
9 is a control group (control) treated with C2C12 DMSO, a negative control group (atrophy) treated with Dexamethasone to induce muscle atrophy, and an experimental group (total ext) .) is a graph that quantifies and compares the measured diameter of each myotube cell with a photograph observing the shape, confirming the effect of improving muscle atrophy induced by Dexamethasone of Bangui extract.
10 is a view confirming the effect of improving the muscular atrophy induced by Dexamethasone of the Bangui extract fractions I to XVII, and confirming that the effect of improving the muscular atrophy induced by Dexamethasone in fractions III, IV and VII to X is the best. am.
11 is a view confirming the effects of compounds 1 to 7 obtained by performing Preparative HPLC on the VI to VIII, and X fractions of the Bangui extract, the roots induced by Dexamethasone in Compound 2 (sinomenine) and Compound 5 (acutumine) It is a drawing confirming that the effect of atrophy improvement is the best.
12 is a control (control) treated with DMSO to C2C12, a negative control group (atrophy) treated with Dexamethasone to induce muscle atrophy, and myotube cells treated with Compound 2 or 5 in myotube cells treated with Dexamethasone to induce muscle atrophy. It is a photograph observing the shape of
13 is a view comparing the measurement of myotube cell diameters in a control group, a negative control group, and an experimental group treated with Compound 2 or 5;
14 is a control group (control) treated with DMSO in C2C12, a negative control group (atrophy) treated with Dexamethasone to induce muscle atrophy, and a Bangui extract obtained by using different solvents in myotube cells in which muscle atrophy was induced by treatment with Dexamethasone. It is a photograph observing the morphology of the treated myotube cells.
15 is a graph comparing the diameters of myotube cells of the control group, the negative control group, and the experimental group treated with the Bangui extract obtained by changing the solvent. From the Bangui extract obtained by adding 0.5% acetic acid to 75% ethanol It is a drawing confirming that the effect of atrophy improvement is the most excellent.
16 is a view confirming that the expression levels of p-p70S6K and p-mTOR proteins related to muscle hypertrophy in the experimental group treated with Compound 2 or 5 are increased by Western blotting.

As a result of intensive research to search for ingredients derived from natural products that can alleviate skeletal muscle reduction, the present inventors confirmed that it effectively prevented the decrease in the diameter of C2C12 cells by dexamethasone in the group treated with the Bangui extract. , to identify its active ingredient and provide it for the use of improvement, prevention, and treatment of muscular atrophy.

In order to trace and separate the active ingredients of Bangui, it was extracted with 50% ethanol, and under the conditions of n-butanol-acetonitrile-water (10:2:8, v/v, 0.5% triethylamine added) Centrifugal partition chromatography was performed to obtain 17 fractions. When each fraction was treated with the weight ratio obtained, activity was shown in six fractions. These active fractions were separated and purified by preparative HPLC to identify their structures through NMR and ESI-mass. Magnoflorine (1), sinomenine (2), liriodentrin (3), N-feruloyltyramine (4), acutumine (5), dauricumine (6), and syringin (7) were identified in the active fraction, and compounds 1-7 were each identified. When 0.1 or 1 nM of dexamethasone-induced skeletal muscle reduction C2C12 cells were treated, the efficacy of compounds 2 and 5 to improve the reduction in skeletal muscle cell thickness was confirmed.

In addition, as a result of performing an extraction experiment by changing the composition of the solvent to confirm the activity-oriented extraction method, the improvement effect on muscle atrophy was the best in the extract obtained by adding 0.5% acetic acid to 75% ethanol. The contents of compounds 2 and 5 were 32.94 and 10.21 mg, respectively.

In the present invention, Banggi ( Sinomenium acutum ) is a creeping stem and rhizome of a banyan tree or a similarly related plant of the Buttercup. It is also called 瓜防己). Since sinomenine and acutumine, which are ingredients that prevent, treat, or improve muscle atrophy, are contained in the whole Bangui tree, the scope of the patent is not limited to the stem and stem bark, but also the root and root bark, leaves, branches, fruits, buds, pollen, etc. Include all of the included Bangui trees. Preferably, it can be obtained by extracting one or two or more together.

One of the muscular atrophy is disuse atrophy of muscles, which includes a decrease in the thickness of the muscle due to activity restrictions due to aging or disease, long bedtime, or use of casts or Gips. . The Bangui extract and sinomenine and/or acutumine component of the present invention can be used for the purpose of helping the prevention of muscle atrophy caused by activity restrictions caused by aging, disease, and the like.

In addition, the insoluble muscle atrophy (disuse atrophy) is accompanied by various clinical diseases such as peripheral nerve damage, cancer and sepsis, as well as long-term bed rest, and aging process. In general, muscle atrophy is accompanied by physiological, histochemical, and biochemical changes due to a decrease in muscle protein and may lead to intrinsic dysfunction of skeletal muscle. It can be used for the purpose of maintaining and preserving the intrinsic function of skeletal muscle resulting from muscle atrophy from the function of protecting muscle fibers.

One of the causes of muscle atrophy may be the absence of mechanical stimulation and denervation of skeletal muscles. The Bangui extract and sinomenine and acutumine components can be used for the purpose of helping prevent and improve symptoms of muscle atrophy that may be caused by it.

In denervated muscle cells, mitochondrial quantitative decrease and dysfunction occur, mitochondrial reactive oxygen species (ROS) production increases, and muscle cell apoptosis due to oxidative damage increases. muscle atrophy is caused

One of the symptoms of cachexia, a highly debilitating symptom of cancer, tuberculosis, and hemophilia, is rapid muscle contraction. can be used for this purpose.

On the other hand, muscular dystrophy (muscular dystrophy) is a disease whose main symptoms are muscle atrophy and muscle weakness caused by the absence of dystrophin-glycoprotein complex formation, which is a component of the striated muscle plasma membrane due to gene mutation. It can be used for the purpose of helping to prevent muscle atrophy and improve symptoms.

In addition, cortisol (cortisol) is a substance that can be secreted in the body due to various acute stresses, and serves to supply energy against stress, but also causes muscle atrophy of the body according to its mechanism. The Bangui extract, sinomenine, and acutumine may be used for the purpose of helping to prevent and improve symptoms, respectively, of muscle atrophy that may be caused by cortisol.

In addition, synthetic corticosteroids may cause steroid induced atrophy, muscle parenchymal damage, etc. as an adverse reaction. The Bangui extract, sinomenine and acutumine may be used for the purpose of helping to prevent and improve symptoms of such steroid muscular atrophy, respectively.

The Bangui extract of the present invention can be extracted according to a conventional method known in the sugar leaf system for extracting an extract from a natural product, that is, using a conventional solvent under conditions of conventional temperature and pressure. For example, in the present invention, the Bangui extract is selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixed solvent thereof. Extraction may be performed using one or more solvents, and extraction may be performed using a mixed solvent of water and an alcohol having 1 to 4 carbon atoms, and in this case, water and alcohol are mixed in a volume ratio of 1:5 to 1:1. It may be a mixture, but more preferably 75% alcohol (ethanol) may be used for extraction. Depending on the mixing ratio, the extraction efficiency of the effective substance having the effect of improving the muscular atrophy of Bangui may vary.

In addition, the method of extracting the extract from the banggi can be extracted through various methods such as hot water extraction, cold extraction, reflux extraction, ultrasonic extraction, but is not limited thereto, and preferably a reflux extraction method can be used.

The prepared extract may then be filtered or concentrated or dried to remove the solvent, and all of filtration, concentration and drying may be performed, and the filtration, concentration or drying process may be performed several times. For example, the filtration may be performed using a filter paper or a reduced pressure filter, the concentration may be performed by a vacuum concentrator, and the drying may be performed by a spray drying method, a freeze drying method, and the like, but is not limited thereto.

In addition, the extract extracted with the solvent is then subjected to a fractionation process with a solvent selected from the group consisting of butanol, n-hexane, methylene chloride, acetone, ethyl acetate, ethyl ether, chloroform, water, and mixtures thereof. , but not limited thereto.

In addition, in the present invention, when extracting the Bangui extract, an edible acid may be added to the solvent to extract, preferably acetic acid, citric acid, malic acid ), succinic acid, fumaric acid, tartaric acid, amino acid, and at least one acid selected from the group consisting of lactic acid may be added to the solvent, Preferably, acetic acid may be added, and in this case, 0.1 to 1 wt%, preferably 0.5 wt%, of acetic acid added to the solvent may be added.

Accordingly, the present invention comprises the steps of (a) reflux extraction after adding an extraction solvent to Banggi; And (b) provides a method for producing a Bangui extract for improving, preventing, or treating muscular atrophy comprising the step of lyophilizing after concentrating the extract extracted from the above under reduced pressure.

In the present invention, muscle atrophy to be improved, prevented, or treated is insoluble muscle atrophy (disuse atrophy of muscles), absence of mechanical stimulation and denervation of skeletal muscle, cachexia, drug-induced muscular atrophy (Drug) induced muscle atrophy), malnutrition induced muscle atrophy, and muscular dystrophy.

The Banggi ( Sinomenium acutum ) extract and sinomenine, a composition for preventing, treating or improving muscle atrophy, comprising an acutumine component, has the effect of preventing, treating or improving muscle atrophy for reduction of muscle fiber bundles, inflammatory cell infiltration of muscles and local fibrosis it could be

The pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions.

In addition, according to the present invention, the pharmaceutical composition for the prevention or treatment of muscular atrophy comprising the Bangui extract, cynomenin, and/or acutumin is powder, granule, tablet, capsule, suspension, emulsion, It can be formulated and used in the form of external preparations such as syrups and aerosols and sterile injection solutions, preferably creams, gels, patches, sprays, ointments, warning agents, lotions, liniment agents, pasta agents, or cataplasma agents. It may have a formulation. Carriers, excipients and diluents that may be included in the composition comprising the extract of Hansokdan include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral use include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients, for example, wetting agents, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As a base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The preferred dosage of the pharmaceutical composition of the present invention varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the pharmaceutical composition of the present invention is preferably administered at 0.0001 to 100 mg/kg per day, preferably 0.001 to 10 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition according to the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes such as parenteral and oral administration, and all modes of administration can be expected, for example, oral, It may be administered by rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

In addition, the oral dosage form may vary depending on the patient's age, sex, and weight, but an amount of 0.1 to 100 mg/kg may be administered once to several times a day. In addition, the dosage may be increased or decreased according to the route of administration, the degree of disease, sex, weight, age, and the like. Accordingly, the above dosage does not limit the scope of the present invention in any way.

In addition, the present invention provides a food composition for improving muscle atrophy and/or muscle loss, comprising the Bangui extract or cynomenin and/or acutumin obtained therefrom as an active ingredient. In addition, Bangui extract, cynomenin, and/or acutumin may be added to food for the purpose of preventing or delaying muscle atrophy and/or muscle loss. When the Bangui extract, cynomenin, and/or acutumin of the present invention is used as a food additive, the Bangui extract, cynomenin, and/or acutumin may be added as it is or used together with other foods or food ingredients, and conventional It can be used appropriately depending on the method. The mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, in the production of food or beverage, the Bangui extract, cynomenin, and/or acutumin of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term intake for health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

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

The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, as in a conventional beverage. The above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as taumartin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, preferably about 0.04-0.10 g per 100 mL of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, Carbonating agents used in carbonated beverages, etc. may be contained. In addition, the composition of the present invention may contain the pulp for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.

As used herein, the term “prevention” refers to any action that suppresses or delays the onset of muscle atrophy or muscle loss by administration of the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which the symptoms of muscular atrophy are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.

As used herein, the term “improvement” refers to any action that reduces a parameter related to muscle atrophy or muscle loss, for example, the severity of symptoms by administration of the composition according to the present invention.

The present invention can apply various transformations and can have various embodiments. Hereinafter, specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention. In describing the present invention, if it is determined that a detailed description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.

[Preparation of Experiment]

1. Experimental materials

The Banggi used in this experiment is based on the characteristics of the Korean Pharmacopoeia (KP) and is a product of Daemyung Pharmaceutical. It was made in China and was manufactured in May 2017 and purchased at the Gyeongdong Market in Seoul. The specimen (HYUP-SA-001) was stored in the Pharmacy Laboratory of Hanyang University College of Pharmacy.

2. Instruments and reagents

2-1. Physicochemical identification and separation and purification

[reagent]

Ethanol, n -butanol, acetonitrile, isopropanol, water (Daejeong Hwageum), methanol (Samjeonsunyak) acetic acid (Merk), triethylamine, ammonium acetate (Sigma aldrich), methanol-d ₄ (Merck), D ₂ O, CDCl ₃ , DMSO-d ₆ (Cambridge isotope)

[device]

HPLC System for Analysis

Agilent 1260 HPLC system (Agilent Technologies, Waldbronn, Germany)

G1312C 1260 Binary Pump, G1322A 1260 degasser, G1329B autosampler, G1316A column oven, G1315D DAD detector, software : Chemstation

HPLC column: HECTOR C18 (5 μm, 4.6 x 250 mm, RS tech corporation, Korea), Capcell pak C18 UG120 (5 μm, 4.6 x 250 mm, Shesiedo, Japan)

HPLC System for Separation

Preparative HPLC (Gilson, France): 321 pump, 157 UV-VIS detector, software: N2000 chromatostasis ver. 3.55

Preparative HPLC column: Hector C18 (5 μm, 20 x 250 mm, RS tech corporation, Korea)

SCPC-100+1000, Spot prep II HPLC instrument

Automatic HPLC system: digital screen, pump, injector, fraction collector, UV/Vis detector, software: SCPC-100+1000 and Spot prep II system Armen instrument

Rotary vacuum evaporator : SB-1200 (Eyala, China)

Low temperature circulator : CCA-111 (Eyala, China)

2-2. Evaluation of the improvement effect of skeletal muscle cell atrophy

[reagent]

High glucose DMEM, fetal bovine serum, horse serum, penicillin-streptomycin solution (Hyclone), trypsin EDTA (Gibco), dimethyl sulphoxide hybri-MAX® dexamethasone (Sigma aldrich)

[device]

Real-Time cell history recorder - JuLi ^™ Stage (NanoEn Tek)

[cell line]

C2C12 (mouse myoblast cell line, Center for Biological Resources, Korea Research Institute of Bioscience and Biotechnology)

3. Extraction and Isolation of Bangui

3-1. Extraction of Bangui

Using 50% ethanol as an extraction solvent, 1.49 kg of Bangui was extracted under reflux cooling for 2 to 4 hours at 5 L each. The extraction solvent was removed using a vacuum concentrator and a freeze dryer.

3-2. Centrifugal partition chromatography (CPC)

In separation using CPC, an ascending mode was used in which the upper layer was a mobile phase and the lower layer was a stationary phase. First, the main peak in the chromatogram (Fig. 2) of the Bangui ethanol extract Based on ad, it was attempted to establish solvent conditions for component separation using CPC.

The solvent is 0.1% formic acid added to water and acetonitrile. 0-5 minutes: 5-8%, 5-15 minutes: 8-10%, 15-20 minutes: 10-15% , 20-25 minutes: 15-20%, 25-30 minutes: 20-80%, 30-40 minutes: 80-90% Gradient elution was performed to increase the composition of acetonitrile. HECTOR C18 (5 μm, 4.6 x 250 mm) was used for the column, and the detection wavelength was 265 nm.

Solvent composition (v/v/v) Triethylamine (%) solvent ratio K value* a b c d n -butanol : acetonitrile : water 0.1 8:2:10 0.140 0.178 0.285 0.322 9: 1: 10 0.195 0.117 0.221 0.336 0.5 8:2:10 0.572 0.173 0.335 0.419 9: 1: 10 0.506 0.106 0.270 0.408 n -butanol : isopropanol : water 0.1 7:3:10 0.196 0.156 0.278 0.415 8:2:10 0.158 0.108 0.186 0.416 9: 1: 10 0.168 0.098 0.136 0.383 0.5 7:3:10 3.950 0.170 0.496 0.733 8:2:10 4.336 0.126 0.555 1.038 9: 1: 10 5.601 0.130 0.578 0.884

Table 1 is a list of solvent compositions used to find the optimal K value for separation of Bangui extract in CPC using a biphasic solvent system (FIG. 1) ( K value = peak area of upper phase/peak area of lower phase). The composition of the optimized solvent referring to the values in the table is n -butanol : acetonitrile : water = 10 : 2 : 8 (v/v/v, 0.5% trietylamine added). This was prepared so that the upper layer and the lower layer were about 4 L each. The upper layer was used as the mobile phase and the lower layer as the stationary phase. First, 1 L of the lower layer, which is the stationary phase, was filled in the rotor, rotated and fixed by centrifugal force. When the upper layer was poured, the lower layer remaining in the rotor was 530 mL. The rotor speed was 1000 rpm, and the initial pressure was 42 bar. Detection wavelengths were measured at 280 and 254 nm. The mobile phase was separated by flowing at a rate of 10 mL/min in Ascending mode. From 4 hours, the experiment was performed by changing to descending mode.

3-3. Preparative HPLC

In order to separate the active ingredient from the six active fractions, prep. HPLC was performed. Prep. HECTOR C18 (5 μm, 20 x 250 mm) was used as the column. Analysis was performed at detection wavelengths of 265 and 253 nm at a flow rate of 10 mL/min, and methanol was separated as a mobile phase by 5-90% gradient elution for 90 minutes.

4. Evaluation of the effect of improving muscle atrophy using C2C12

After differentiation of C2C12 mouse myoblasts (myoblasts) into myotubes (myotube cells), 1 μM of dexamethasone was treated for 48 hours to induce atrophy, and using this, extracts, fractions, and compounds each were atrophy. It was used to evaluate the improvement effect.

4-1. C2C12 cell preparation

The cell line used in the experiment is the C2C12 mouse myoblast cell line. Cell lines obtained from the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center, subcultured several times, and cryopreserved were used. The composition of cryopreserved cell lines was 20% FBS and 10% DMSO, and HyClone ^™ DMEM/high glucose was used as the culture medium. 1.5 2.0 x 10 ⁶ cells per 1 mL of cryopreservation medium were aliquoted into freezing tubes. This was frozen for 24 hours in a deep freezer at -80°C in an isopropanol tank and stored in liquid nitrogen.

Thawed cell lines are passaged at about 90% full of 100 mm culture dish area every 2 days. This was repeated 3 times to induce division for a total of 6 days and then used for seeding. For seeding, the amount of division medium was 1 mL for each well of a 12-well plate and the number of cells was 0.10.15 x 10 ⁶ cells. Fragmentation was carried out for 2 days to about 90% or more fullness. The composition of the cleavage medium is 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin using HyClone ^™ DMEM/high glucose as a culture medium.

When each well of the 12 well plate is more than 90% full, the differentiation medium is processed. The differentiation medium was replaced every 2 days, and 1 mL each was treated 3 times, for a total of 6 days. The composition of the differentiation medium is 2% HS (horse serum) and 1% penicillin-streptomycin using HyClone ^™ DMEM/high glucose as a culture medium. After the C2C12 myotube was sufficiently differentiated, it was treated with a serum-free culture medium for 12 hours. Serum free is HyClone ^™ DMEM/high glucose that does not contain serum such as FBS or HS, and only 1% penicillin-streptomycin is added. The preparation stage of myotube for evaluating the improvement of muscle atrophy is up to the treatment of serum-free culture medium.

4-2. Active search using C2C12 myotube

Samples were dissolved in DMSO, diluted in serum-free medium, and treated according to a set concentration. Since DMSO is cytotoxic, it was not allowed to exceed 10% of the final volume. For example, when the final drug concentration was 1 nM, 1 μL of a sample of 1 μM concentration was treated in 1 mL serum free culture medium. The control group is treated with only DMSO, the vehicle, and the atrophy group is treated with a final concentration of 1 μM of dexamethasone. Samples were evaluated by treatment with 1 μM of dexamethasone for 48 hours.

4-3. Determination of C2C12 myotube diameter

After 48 hours of sample treatment, cell photos were taken at 10x magnification using a JuLi ^™ Stage. 3 points per well of 12 well plate were taken in 2 x 2 stich mode, and 50 myotube diameters were measured per cell picture using the 'Straight' tool of ImageJ program for each stiched picture and averaged, each 150 myotubes per well were counted. The scale bar when stitched to 2 x 2 was 500 μm. When the length of the scale bar of the stitched photo was measured with the 'Straight' tool of ImageJ, it was 560 μm. Converting the measured value to the actual length using these two was obtained by multiplying 500/560 (about 0.893).

4-4. statistical analysis

The error of the graph measuring the C2C12 diameter was expressed as the standard error. One-way ANOVA was used for comparison of each group ( ^* p <0.05, ^** p <0.01, ^*** p <0.005 vs. control / ^# p <0.05, ^## p <0.01, ^### P <0.005 vs. atrophy).

5. Extraction method to increase the yield of active ingredients

By adding water to ethanol, extraction yields were compared with the following solvents. Extracted with 0, 25, 50, 75, 100% ethanol with 0.5% acetic acid and a solvent without addition.

6. Confirmation of expression level of muscle atrophy and hypertrophy related proteins

Each of the cells previously photographed was measured for protein activity (Atrogin-1, MuRF-1, p-mTOR, and p-p70S6K) using Western blotting as a basic protocol.

By measuring the expression levels of Atrogin-1 and MuRF-1 in the muscular atrophy pathway and p-mTOR and p-p70S6K in the hypertrophic pathway, the biochemical activity of Bangui extract and sinomenine and acutumine was verified.

[Experiment result]

1. Extraction and Isolation of Bangui

1-1. extraction

After extracting 1.49 kg of pulverized Bangui twice and freeze-drying, the weight of the crude extract was 221.4 g. The yield at this time is 14.81%.

1-2. Separation and purification using CPC and prep HPLC

17 fractions were obtained using CPC (FIG. 4), and the weight and chromatogram of each fraction are shown in FIG. When the muscle atrophy effect of each fraction was evaluated, there was an effect of improving muscle atrophy in fractions III, IV and VII to X. Therefore, in order to find an active substance in these active fractions, it was purified using preparative HPLC, and compounds 1 to 7 were obtained. The effect of the CPC fraction was shown in Preparation 3-2 of the above experiment, and the effect of compounds 1 to 7 was shown in Preparation 3-3 of the above experiment. The solvent conditions, column, and detection wavelength are as shown in FIG. 2 .

1-3. Structural identification of isolated substances

Compounds 1-7 were obtained by prep HPLC purification of fractions III, IV, IX containing the main compound peak and fractions VI-VIII and X, which showed improvement in muscle atrophy compared to the atrophy group. The chromatogram of the compound is shown in FIG. 7 . NMR and MS spectrum used for structural identification are shown below.

The solvent is 20 mM ammonium acetate added to water and methanol. 0~15 min: 15~30%, 25~40 min: 30~60%, 40~45 min: 60 -80%, gradient elution was performed with increasing methanol composition. HECTOR C18 (5 μm, 4.6 x 250 mm) was used for the column, and the detection wavelength was 265 nm.

Magnoflorine (Compound 1)

C ₂₀ H ₂₄ NO ₄ ⁺ , ESI-MS (negative) 342 m/z [MH] ^-

¹ H-NMR (D ₂ O, 400 MHz): δ 6.62 (d, J = 8.0 Hz, 1H, H-9), 6.39 (s, 1H, H-3), 6.33 (d, J = 8.0 Hz, 1H, H-8), 3.78 (s, 3H, -OCH ₃ ), 3.66 (s, 3H, -OCH ₃ ), 3.23 (m, 1H, H-5), 2.98 (s, 3H, N ⁺ -CH ₃ ), 2.93-2.80 (m, 1H, H-7), 2.65 (d, J = 9.6 Hz, 2H, H-6a), 2.47 (s, 3H, N ⁺ -CH ₃ ), 2.36 (m, 1H) , H-7), 1.90 (t, J = 13.1 Hz, 1H, H-7a).

¹³ C-NMR (D ₂ O, 100 MHz): δ 151.79 (C-2), 150.29 (C-10), 148.75 (C-1), 147.75 (C-11), 125.79 (C-7a), 122.32 (C-8), 121.93 (C-8a), 120.52 (C-11a), 117.47 (C-1a), 116.72 (C-1b), 111.30 (C-9), 109.80 (C-3), 69.72 ( C-6a), 61.61 (C-5), 56.59 (-OCH ₃ ), 56.25 (-OCH ₃ ), 54.06 (N ⁺ -CH ₃ ), 43.38 (N ⁺ -CH ₃ ), 30.51 (C-7) , 23.98 (C-4).

Sinomenine (Compound 2)

C ₁₉ H ₂₃ NO _{4 ,} ESI-MS (positive) 330 m/z [M+H] ⁺

¹ H-NMR (CDCl ₃ , 400 MHz): δ 6.63 (d, J = 8.3 Hz, 1H, H-1), 6.53 (d, J = 8.2, 1H, H-2), 5.46 (d, J = 2.2 Hz, 1H, H-8), 4.34 (d, J = 15.6 Hz, 1H, H-5b), 3.80 (s, 3H, -OCH ₃ ), 3.48 (s, 3H, -OCH ₃ ) _, 3.09 ( m, 1H), 2.88 (m, 1H), 2.44 (d, J = 15.6 Hz, 1H), 2.43 (s, 3H, N-CH ₃ ), 2.44 (d, J=15.6 Hz, 1H, H-5a ), 2.08 (m, 1H), 1.92-1.88 (m, 1H).

¹³ C-NMR (CDCl ₃ , 100 MHz): δ 194.10 (C-6), 152.51 (C-7), 145.09 (C-3), 144.82 (C-4), 130.44 (C-11), 122.67 ( C-12), 118.39 (C-1), 115.14 (C-8), 109.12 (C-2), 56.85 (C-9), 56.21 (-OCH ₃ ), 54.93 (-OCH ₃ ), 49.31 (C -5), 47.27 (C-16), 45.97 (C-14), 42.86 (N-CH ₃ ), 40.59 (C-13), 36.10 (C-15), 24.40 (C-10).

Liriodendrin (Compound 3)

C ₃₄ H ₄₆ O _18, ESI-MS (positive) 743 m/z [M+H] ⁺

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 6.66 (s, 4H, H-2, 2', 6, 6'), 4.95 (m, 4H, H-3, 3', 5, 5 '), 4.67 (d, J = 3.7 Hz, 2H, H-7, 7'), 4.20 (dd, J=8.8, 6.5 Hz, 2H, H-8, 8'), 3.10 (m, 2H, H -9, 9').

¹³ C-NMR (DMSO-d ₆ , 101 MHz): δ 152.61 (C-3, 3', 5, 5'), 137.09 (C-4, 4'), 133.71 (C-1, 1'), 102.66 (C-2, 2', 6, 6'), 85.06 (C-7, 7'), 74.15 (C-8, 8'), 56.42 (-OCH ₃ ), 53.60 (C-9, 9') ), 104.23 (C-1"), 77.21 (C-3"), 76.51 (C-5"), 71.34 (C-2"), 69.92 (C-4"), 60.91 (C-6").

N -feruloyltyramine (compound 4)

C ₁₈ H ₁₉ NO _{6 ,} ESI-MS (positive) 314 m/z [M+H] ⁺

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ7.30 (d, J = 15.7 Hz, 1H, H-2'), 7.11 (d, J = 2.0 Hz, 1H, H-5'), 7.01 (d, J = 8.4 Hz, 2H, H-4, 8), 6.97 (dd, J = 8.2, 2.0 Hz, 1H, H-9'), 6.78 (d, J = 8.1 Hz, 1H, H- 8'), 6.68 (d, J = 8.4 Hz, 2H, H-5, 7), 6.42 (d, J = 15.7 Hz, 1H, H-6'), 3.80 (s, 3H, -OCH ₃ ), 2.64 (d, J = 7.4 Hz, 2H, H-2).

¹³ C-NMR (DMSO-d ₆ , 100 MHz): δ 165.24 (C-1), 155.60 (C-6), 148.19 (C-6'), 147.78 (C-7'), 138.80 (C-3) '), 129.50 (C-3), 129.43 (C-4, 8), 126.40 (C-4'), 121.46 (C-9'), 119.02 (C-2'), 115.61 (C-8') , 115.08 (C-5, 7), 110.72 (C-5'), 55.50 (-OCH ₃ ), 40.63 (C-1), 34.41 (C-2).

Acutumine (Compound 5)

C ₁₉ H ₂₄ ClNO _{6 ,} ESI-MS (positive) 398 m/z [M+H] ⁺

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 6.18 (d, J = 6.1 Hz, 1H, H-3), 5.43 (d, J = 0.6 Hz, 1H, H-1), 4.47 (dd , J = 12.0, 6.9 Hz, 1H, H-10), 3.98 (s, 3H, -OCH ₃ ), 3.86(s, 3H, -OCH ₃ ), 3.72 (d, J = 11.1 Hz, 1H, H- 5b), 3.55 (s, 3H, -OCH ₃ ), 2.60-2.66 (m, 1H, H-15b), 2.47 (d, J=12.5 Hz, 1H, H-16), 2.35 (s, 1H, H) -16), 2.41 (d, J=10.4 Hz, 1H, H-15a), 2.20-2.24 (m, 1H, H-14b), 2.13 (td, J=11.1, 6.4 Hz, 1H, H-5) , 1.50 (dd, J=11.4, 4.1 Hz, 1H, H-14a).

¹³ C-NMR (DMSO-d ₆ , 100 MHz): δ201.07 (C-4), 192.58 (C-6), 189.03 (C-2), 159.88 (C-8), 138.31 (C-7) , 105.40 (C-3), 72.36 (C-13), 69.84 (C-1), 67.57 (C-11), 60.76 (C-19), 60.28 (C-18), 59.50 (C-17), 57.37 (C-10), 52.89 (C-12), 51.51 (C-15), 46.23 (C-5), 40.93 (C-9), 38.04 (C-14), 36.53 (C-16).

Dauricumine (Compound 6)

C ₁₉ H ₂₄ ClNO _{6 ,} ESI-MS (positive) 398 m/z [M+H] ⁺

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 5.41 (s, 1H, H-3), 4.56 (s, 1H, H-1), 4.54 (dd, 1H, H-10), 3.97 ( s, 3H, -OCH ₃ ), 3.86 (s, 3H, -OCH ₃ ), 3.54 (s, 3H, -OCH ₃ ), 3.17 (s, 3H, -OCH ₃ ), 2.78 (dd, J = 10.2, 6.2 Hz, 1H, H-5b), 2.43 (dd, 1H, H-15a), 2.27 (dd, J = 12.6, 7.2 Hz, 1H, H-9a), 2.18 (s, 1H, H-14b), 1.99 (td, J = 11.6, 6.3 Hz, 1H, H-14a).

¹³ C-NMR (DMSO-d ₆ , 100 MHz): δ199.73 (C-4), 190.60 (C-6), 188.48 (C-2), 135.34 (C-7), 104.93 (C-3) , 69.88 (C-13), 67.20 (C-1), 60.55 (C-11), 59.44 (C-19), 58.95 (C-18), 55.82 (C-17), 52.19 (C-10), 46.59 (C-12), 43.96 (C-15), 40.73 (C-5), 40.43 (C-9).

Syringin (Compound 7)

C ₁₇ H ₂₄ O _9, ESI-MS (positive) 372 m/z [M+H] ⁺

¹ H-NMR (DMSO-d ₆ , 400 MHz): δ 6.72 (s, 2H, H-2, 6), 6.46 (d, J = 15.9 Hz, 1H, H-7), 6.33 (dt, J = 15.9, 5.0 Hz, 1H, H-8), 4.91 (d, J = 7.2 Hz, 1H, H-1'), 4.10 (d, J = 4.3 Hz, 2H, H-6'), 3.77 (s, 6H, -OCH ₃ ), 3.58 (d, J = 11.2 Hz, 1H, H-9), 3.40 (dd, J = 11.8, 5.5 Hz, 1H, H-9), 3.20 (d, J = 2.2 Hz, 1H, H-2'), 3.18 (s, 1H, H-3'), 3.17 - 3.1 0(m, 1H, H-5'), 3.06 -2.99 (m, 1H, H-4').

¹³ C-NMR (DMSO-d ₆ , 100 MHz): δ 152.69 (C-3, 5), 133.84 (C-4), 132.57 (C-1), 130.16 (C-8), 128.42 (C-7) ), 104.45 (C-2, 6), 102.54 (C-1'), 77.21 (C-5'), 76.54 (C-3'), 74.17 (C-2'), 69.92 (C-4') , 61.45 (C-6'), 60.88 (C-9), 56.34 (-OCH ₃ ).

2. Effect of improving skeletal muscle cell atrophy

2-1. Effects of Bangui Extract

When treated with Bangui extract (30 g/mL). Compared to the atrophy group treated only with dexamethasone, the improvement of muscle atrophy was 13.3%. The average myotube diameter of the control group was around 18-23 μm, and the atrophy group treated with dexomethasone 1 M was around 14-18 μm. Referring to FIG. 9 , the atrophy group showed a significant muscle atrophy rate of 22.5% compared to the control group. At this time, the group treated with 30 μg/mL of Bangui 50% ethanol extract showed a significant improvement in muscle atrophy by 13.3% compared to the atrophy group.

2-2. Effect of CPC fraction

Treatment concentrations of each fraction are summarized in Table 2 below. 10 is a result of processing 17 fractions obtained through CPC. The treatment concentration of each fraction was calculated as a weight ratio based on the treatment of 30 μg/mL of the crude extract. As shown in FIG. 10, the improvement rate was the best in the fractions of III, IV and VII-X.

2-3. Effect of Separated Substances

The effects of compounds 1 to 7 isolated through Prep HPLC were evaluated at 0.1 and 1 nM ( FIG. 11 ). As a result, sinomenine (Compound 2) and acutumine (Compound 5) showed significant improvement rates, and the effects of the compounds were evaluated again.

The improvement effect on muscle cell atrophy was evaluated by re-treatment with sinomenine (2) or acutumine (5), two compounds whose activity was confirmed ( FIG. 13 ). Each compound was treated with 0.1 and 1 nM as in the previous experiment. Compared to the control group, the atrophy group showed significant muscle atrophy at 20.9%, and at concentrations of 0.1 and 1 nM, 9.9, 9.3% of sinomenine and 7.4, 11.3% of acutumine showed significant improvement compared to the atrophy group. Their cell pictures are shown in FIG. 12 .

3. Effect of Extraction Method and Extraction by Solvent to Increase the Yield of Active Substances

3-1. Active Substance Yield Increase Extraction Method

As confirmed in the experimental results 2-3, the components of the Bangui component having the effect of improving skeletal muscle cell atrophy are sinomenine (2) and acutumine (5). These are all alkaloid compounds containing nitrogen. Therefore, in order to increase the ratio of these components in the extract, in the expectation that the extraction efficiency will vary depending on the presence or absence of acidity of the solvent, the extracts extracted under acidic or non-acidic conditions and quantified are summarized in Table 3 below. Acidic conditions are 0, 25, 50, 75, and 100% ethanol solvents each added with 0.5% acetic acid.

% Ethanol 0.5% Acetic acid compound mg/extract g compound 2 compound 5 0 - 22.86 6.21 + 21.76 7.24 25 - 27.99 7.98 + 24.54 7.61 50 - 20.98 9.16 + 21.99 7.04 75 - 29.52 10.87 + 32.94 10.21 100 - 38.61 13.38 + 37.85 13.44

3-2. Improvement of muscle atrophy of extracts by solvent

The improvement of muscle atrophy of acidic and non-acidic condition extracts was evaluated. Looking at the graph of FIG. 15, the group in which 0.5% acetic acid was added to 75% ethanol showed the highest and most significant improvement in muscle atrophy compared to the atrophy group. Their cell pictures are shown in FIG. 14 .

4. Effect of regulating skeletal muscle cell protein expression level

In Example 2-3, the improvement of muscle atrophy of Sinomenine and Acutumine was confirmed, and then, Western blotting was performed to confirm the effect of each compound on the muscle atrophy and hypertrophy pathways. As a result, it was confirmed that the expression levels of Atrogin-1 and MuRF-1 involved in muscle atrophy were not affected by treatment with Sinomenine or Acutumine, but the expression levels of p-mTOR and p-p70S6K involved in muscle hypertrophy were increased. (Fig. 16).

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (13)

Banggi ( Sinomenium acutum ) A pharmaceutical composition for preventing or treating muscle atrophy comprising an extract as an active ingredient.
According to claim 1,
The Bangui extract is at least one selected from the group consisting of water, C ₁ to C ₄ lower alcohol, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixture thereof. A pharmaceutical composition, characterized in that extracted with a solvent.
3. The method of claim 2,
The Bangui extract is acetic acid, citric acid, malic acid, succinic acid, fumaric acid, tartaric acid, and amino acids in the solvent. A pharmaceutical composition, characterized in that extracted with a solvent further comprising at least one acid selected from the group consisting of lactic acid.
The method of claim 1,
The muscle atrophy is at least one selected from the group consisting of insoluble muscle atrophy, muscular atrophy caused by the absence of mechanical stimulation, muscle atrophy due to denervation of the skeletal pole, and a mixture thereof, a pharmaceutical composition .
A pharmaceutical composition for preventing or treating muscle atrophy, comprising at least one selected from the group consisting of sinomenine and acutumine as an active ingredient.
6. The method of claim 5,
The sinomenin and acutumin are Bangui ( Sinomenium acutum ) Characterized in that it is derived from, a pharmaceutical composition.
6. The method of claim 5,
The composition includes cynomenin and acutumin, and 2 to 5 parts by weight of cynomenin based on 1 part by weight of acutumin, a pharmaceutical composition.
Banggi ( Sinomenium acutum ) A food composition for preventing or improving muscle loss comprising an extract as an active ingredient.
9. The method of claim 8,
The Bangui extract is at least one selected from the group consisting of water, C ₁ to C ₄ lower alcohol, n-hexane, ethyl acetate, acetone, butyl acetate, 1,3-butylene glycol, methylene chloride, and a mixture thereof. A food composition, characterized in that it is extracted with a solvent.
9. The method of claim 8,
The Bangui extract is acetic acid, citric acid, malic acid, succinic acid, fumaric acid, tartaric acid, and amino acids in the solvent. A food composition, characterized in that it is extracted with a solvent further comprising at least one acid selected from the group consisting of lactic acid.
A food composition for preventing or improving muscle loss comprising at least one selected from the group consisting of sinomenine and acutumine as an active ingredient.
12. The method of claim 11,
The sinomenin and acutumin are Bangui ( Sinomenium acutum ) Characterized in that it is derived from, a food composition.
12. The method of claim 11,
The composition includes cynomenin and acutumin, and 2 to 5 parts by weight of cynomenin based on 1 part by weight of acutumin, a food composition.

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