KR20240133112A - Composition for preventing, improving or treating muscle sarcopenia-related disease - Google Patents

Abstract

The present invention relates to a pharmaceutical or food composition comprising an extract of Salvia militaris, a fraction of the extract, or an effective ingredient derived from the extract or fraction, which is very useful for effectively preventing, alleviating, improving, or treating diseases related to muscle loss.

Description

Composition for preventing, improving or treating muscle sarcopenia-related disease

The present invention relates to a composition for preventing, improving or treating diseases related to muscle loss, and more particularly, to a composition comprising an extract or fraction of Salvia miltiorrhiza or an effective ingredient derived from such an extract or fraction, which exhibits an effect of preventing, improving or treating diseases related to muscle loss, such as atony, muscular atrophy, sarcopenia, myasthenia, cachexia and muscular dystrophy.

One of the physiological changes that occur in the elderly is sarcopenia, which occurs as muscle mass decreases with age. According to the results of the National Health and Nutrition Survey (IV) in Korea, muscle mass begins to decrease around the age of 40 for men and decreases after the age of 55 for women. In the case of the general population, a new concept of sarcopenic obesity is emerging, in which body composition changes with age, but body fat increases but muscle mass decreases. In addition, lipid consumption and excessive nutrient intake cause fat accumulation, and in particular, fat accumulation in muscles secretes a lot of inflammatory cytokines such as TNF-α (tumor necrosis factor-alpha) and IL-6 (interleukin-6), which indirectly affect protein metabolism and directly affect insulin sensitivity, thereby causing a decrease in muscle mass. It is reported that a decrease in muscle mass causes a vicious cycle of increased body fat by decreasing physical activity and reducing the basal metabolic rate, which is the amount of calories consumed during normal times. This sarcopenia is also reported to be related to the patients' falls, trauma, functional disorders, increased hospitalization rates, decreased quality of life, and increased mortality rates by reducing the ability to respond to external stress due to muscle loss. Sarcopenia is also associated with various diseases, and when considering the age, sex, body mass index, and lifestyle of the subjects of diabetes, which is one of the chronic diseases, the incidence of sarcopenia in diabetic patients was reported to be about three times higher than that in the general population. In addition, another muscle in our body, the cardiac muscle, can also be damaged, and a representative example is myocardial infarction.

Meanwhile, Salvia miltiorrhiza (red sage or Chinese sage) is a perennial medicinal plant belonging to the Labiatae family, and includes Salvia miltiorrhiza Bunge, Salvia bowleyana Dunn, Salvia przewalskii Maxim., Salvia przewalskii Maxim. var. mandar inorum (Diels) Stib., Salvia yunnanensis C. H. Wright], Salvia kiaometiensis Levl., f. pubescens Stib., Salvia miltiorrhiza Bunge var. alba C. Y. Wu et H. W. Li, mss., Salvia digitaloides Diels, Salvia trijuga Diels, and Salvia plectranthoides Girff.) etc. Salvia militaris is called red ginseng (赤參) or red ginseng (丹蔘) because its roots are red. The roots are collected in spring or fall, dried, and used as a medicinal herb. It has a unique smell and a bitter taste.

Prior literature related to sarcopenia includes “Use of Ginkgo Biloba Extract for Preparation of Medicine for Treating Sarcopenia (Korean Patent No. 0891393)”, “Prevention and Treatment of Sarcopenia (Korean Publication Patent No. 2012-0058457)”, “Composition and External Agent for Promoting Muscle Differentiation and Strengthening Muscle (Korean Publication Patent No. 2015-0024586)”, Piwen Zhao et al, Anabolic Effect of the Traditional Chinese Medicine Compound Tanshinone IIA on Myotube Hypertrophy Is Mediated by Estrogen Receptor, Planta Med, 81, 578-585(2015), KA Szczesniak et al, Dietary supplementation of b-hydroxy-b-methylbutyrate in animals-a review, Journal of Animal Physiology and Animal Nutrition 99, pp 405-417(2015), Hongmei Wu et al, Effect of beta-hydroxy-beta-methylbutyrate supplementation on muscle loss in older adults: A systematic review and meta-analysis, Archives of Gerontology and Geriatrics, 61, pp168-175 (2015). However, the above prior literature does not specifically disclose treatment of sarcopenia with Salvia miltiorrhiza extract.

Korean Patent No. 0891393, Use of Ginkgo Biloba Extract for the Preparation of Medicine for Treating Sarcopenia, Registered on March 25, 2009. Korean Patent Publication No. 2012-0058457. Prevention and treatment of sarcopenia, published on June 7, 2012. Korean Patent Publication No. 2015-0024586, Composition and external agent for promoting muscle differentiation and strengthening muscles, published on March 9, 2015.

Hongmei Wu et al, Effect of beta-hydroxy-beta-methylbutyrate supplementation on muscle loss in older adults: A systematic review and meta-analysis, Archives of Gerontology and Geriatrics, 61, pp 168-175, 2015. K. A. Szczesniak et al, Dietary supplementation of b-hydroxy-b-methylbutyrate in animals-a review, Journal of Animal Physiology and Animal Nutrition 99, pp 405-417, published 2015. Piwen Zhao et al, Anabolic Effect of the Traditional Chinese Medicine Compound Tanshinone IIA on Myotube Hypertrophy Is Mediated by Estrogen Receptor, Planta Med, 81, pp 578-585, published 2015.

Against this backdrop, the inventors of the present invention have conducted extensive research to discover the optimal compound that can be used as a material for preventing, improving, or treating various muscle loss-related diseases including sarcopenia, and as a result, they have confirmed that a composition containing an extract or fraction of Salvia miltiorrhiza or an effective ingredient derived from such an extract or fraction exhibits a preventive, improving, or treating effect on muscle loss-related diseases such as atony, muscular atrophy, sarcopenia, myasthenia, cachexia, and muscular dystrophy, thereby completing the present invention.

The purpose of the present invention is to provide a composition, medicine, health functional food or functional food for preventing, improving or treating a disease related to muscle loss, comprising a Salvia miltiorrhiza extract, a fraction thereof or an effective ingredient derived from the Salvia miltiorrhiza extract and/or fraction.

According to one aspect of the present invention for solving the above problem, a pharmaceutical composition or food composition containing a Salvia militaris extract as an effective ingredient for preventing, improving or treating a disease related to muscle loss can be provided. The food composition can be a health functional food.

According to one aspect of the present invention, the disease related to muscle loss may be at least one selected from the group consisting of atony, muscular atrophy, sarcopenia, myasthenia, atonia, cachexia, and muscular dystrophy, and the disease related to muscle loss may be caused by aging, cancer, kidney disease, or diabetes.

According to one aspect of the present invention, the Salvia militaris extract may be obtained by extracting Salvia militaris with water, alcohol, or a mixture thereof. Preferably, it may be an extract using alcohol or hot water, or a combination thereof.

According to one aspect of the present invention, the Salvia militaris extract in the composition may include tanshinone 2a, cryptotanshinone, or a combination thereof. Specifically, the Salvia militaris extract includes a combination of tanshinone 2a and cryptotanshinone, and the weight ratio of tanshinone 2a: cryptotanshinone may be, but is not limited to, 1:1 to 1:5. In addition, the Salvia militaris extract includes a combination of tanshinone 2a and salvianolic acid, and the weight ratio of tanshinone 2a: salvianolic acid may be, but is not limited to, 1:20 to 20:1, preferably 1:10 to 10:1.

According to one aspect of the present invention, the composition may further comprise nicotinamide mononucleotide (NMN).

According to one aspect of the present invention, a food composition for preventing or improving a disease related to muscle loss, a food composition for increasing muscle, a food composition for improving muscle fatigue, or a food composition for improving muscle exercise ability can be provided, the food composition containing a Salvia miltiorrhiza extract as an effective ingredient.

According to one aspect of the present invention, a medium composition for muscle cell differentiation can be provided comprising a Dansam extract, a fraction of the extract, or an effective ingredient derived from the extract or fraction.

According to one aspect of the present invention, a method for muscle cell differentiation can be provided, comprising a step of culturing isolated myoblasts with a Salvia miltiorrhiza extract, a fraction of the extract, or an effective ingredient derived from the extract or fraction.

The pharmaceutical composition comprising the extract of Salvia militaris, fractions thereof and effective ingredients derived therefrom of the present invention is very useful because it can prevent, alleviate, improve or treat diseases related to muscle loss such as sarcopenia.

Figure 1 shows the results of measuring AMPK protein expression in muscle tissue when taking a red ginseng extract.

The present invention is described in more detail below.

The present invention provides a pharmaceutical composition characterized by containing an extract of Salvia militaris as an effective ingredient, a fraction of the extract, or an effective ingredient derived from the extract or fraction. This pharmaceutical composition may be a composition for preventing, improving, or treating a disease related to muscle loss.

Specifically, the muscle-related disease may be at least one selected from the group consisting of atony, muscular atrophy, sarcopenia, myasthenia, atonia, cachexia, and muscular dystrophy, and the muscle wasting-related disease may be caused by, but is not limited to, aging, cancer, or diabetes. In a specific embodiment, the muscle wasting-related disease may be a disease including, but is not limited to, a short physical performance battery (SPPB) as a clinical indicator.

In the present invention, Salvia miltiorrhiza (red sage or Chinese sage) is a perennial medicinal plant belonging to the Labiatae family, and includes Salvia Miltiorrhiza Bunge, Salvia bowleyana Dunn, Salvia przewalskii Maxim., Salvia przewalskii Maxim. var. mandar inorum (Diels) Stib., Salvia yunnanensis C. H. Wright], Salvia kiaometiensis Levl., f. pubescens Stib., Salvia miltiorrhiza Bunge var. alba C. Y. Wu et H. W. Li, mss., Salvia digitaloides Diels, Salvia trijuga Diels, and It may be at least one selected from the group consisting of Salvia plectranthoides Girff.

In one embodiment, the above-mentioned red ginseng extract may contain tanshinone 2a, cryptotanshinone, salvianolic acid B or a combination thereof as effective ingredients.

In the present invention, the term "Salt ginseng extract" means an extract obtained by extracting Salt ginseng. As a specific example, the Salt ginseng extract may be prepared by drying Salt ginseng according to a conventionally known method, cutting or crushing it into a size appropriate for extraction, and extracting it using an appropriate extraction solvent, and may be subjected to pretreatment prior to extraction.

The extraction method may be performed using a known herbal medicine extraction method such as hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction, but is not limited thereto. In addition, the extract may include not only the extract itself, but also a diluted or concentrated extract, and a dried product obtained by drying the extract.

In one embodiment, the Salvia militaris extract of the present invention may be an extract obtained by extracting Salvia with water, alcohol, or a mixture thereof (mixed solvent), an organic solvent (ethyl acetate, dichloromethane, chloroform, hexane, etc.). Preferably, the alcohol may be a lower alcohol having 1 to 4 carbon atoms. In a specific embodiment, the Salvia militaris extract may be an extract obtained by extracting Salvia with water, ethanol, or an ethanol aqueous solution (water:ethanol = 1:10 to 10:1 volume ratio), and may also be an alcohol extract, a hot water extract, or a mixture thereof.

In a specific embodiment, the Salvia militaris extract is a mixture of an alcohol extract and a hot water extract of Salvia militaris, and the weight (or volume) ratio of the alcohol extract: the hot water extract may be 1:10 to 10:1. In one embodiment, the effective ingredients of the Salvia militaris alcohol extract may include Cryptotanshinone and Tanshinone IIa, and the effective ingredient of the Salvia militaris hot water extract may include Salvianolic acid B.

The solvent used during extraction may be used in an amount of 5 to 12 times, 6 to 10 times, or 8 times the weight of the ginseng (preferably dry weight), but is not limited thereto.

As a specific example, the above-mentioned ginseng may be extracted at a temperature of 70 to 100°C, or 80 to 100°C, for 1 to 7 hours, 2 to 6 hours, or 5 hours. This extraction may be performed once to several times, once to three times, or once to twice, and the extracted pretreatment extract may be further filtered, concentrated, and/or dried. The method used at this time may be a filtration, concentration, or drying method commonly used in the production of extracts without limitation.

In the present invention, the term "fraction" means a result obtained by a fractionation method for separating a specific component or a specific group from a mixture containing various components. The fractions of the Salvia miltiorrhiza extract of the present invention can be obtained by suspending the extract concentrate of Salvia miltiorrhiza water, alcohol, or alcohol aqueous solution in water and then sequentially fractionating using a nonpolar solvent such as hexane, ethyl acetate, chloroform, or butanol.

In one embodiment, the amount of the fractionation solvent used is 1 to 5 times, 1 to 10 times, or 1 to 20 times the volume of water, a carbon 1 (C1) to 4 (C4) alcohol, nucleic acid, ethyl acetate, chloroform, butanol, or a mixed solvent thereof, relative to 1 kg of the Dansam extract, and the polar or nonpolar solvent-soluble layer can be extracted and separated 1 to 10 times, preferably 2 to 5 times, to obtain the fractionation. In addition, such solvent fractionation may be performed sequentially.

As an example, the above-mentioned red ginseng extract may be a dried extract (red ginseng dried extract), but is not limited thereto.

The composition of the present invention may contain 0.001 to 90 wt%, 0.01 to 70 wt%, 0.1 to 50 wt%, or 1 to 30 wt% of the extract of Salvia militaris, a fraction of the extract, or an effective ingredient derived from the extract or isolate, based on the total weight of the composition, but is not limited thereto.

The above-mentioned Salvia militaris extract can further include nicotinamide mononucleotide (NMN) and/or alfalfa extract, and a synergistic effect on muscle loss-related diseases was confirmed by mixed use with these. The above-mentioned nicotinamide mononucleotide (NMN) is an intermediate of NAD biosynthesis produced from nicotinamide (NAM) and phosphoribosyl pyrophosphate (PRPP) by nicotinamide phosphoribosyltransferase enzyme (NAMPT). In addition, alfalfa ( Medicago sativa) is a legume and is mainly used as animal feed, and contains abundant high-quality protein and nutrients such as vitamin K and calcium.

In one embodiment, the weight ratio of the above-mentioned Dansam extract:nicotinamide mononucleotide (NMN) may be included in the composition at 1:10 to 10:1, more specifically 1.5:1 to 3:1, and even more specifically 1:1, but is not limited thereto.

In another aspect, the weight ratio of the Dansam extract:Alfalfa extract may be included in the composition at 1:100 to 1:1, more specifically 1:5 to 1:30, and even more specifically 1:10, but is not limited thereto.

The above alfalfa extract can be an extract obtained by collecting alfalfa leaves and using water, C1 to C4 alcohol, an alcohol solution, or an organic solvent (ethyl acetate, chloroform, hexane, dichloromethane, etc.), or by pressing alfalfa leaves and using a sieve to remove the residue.

Alfalfa, also known as Medicago sativa , is a perennial flowering plant in the legume family, and is cultivated as an important forage crop in many countries, including the United States, Canada, Argentina, France, Australia, the Middle East, and South Africa. Medicago, which looks very similar to clover, has been cultivated by humans since at least the 4th century BC, and some use it as a medicinal plant.

The above pharmaceutical composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, solutions, aerosols, etc., external preparations, suppositories, and sterile injection solutions, respectively, according to conventional methods. Carriers, excipients, and diluents that can be included in the above pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrating agents, surfactants, sweeteners, and acidulants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the pharmaceutical composition of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, and acidulants can be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, tween-61, cacao butter, laurin butter, and glycerogelatin.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and body weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. The determination of the dosage based on these factors is within the level of those skilled in the art, and the dosage is generally in the range of 0.01 mg/kg/day to about 50 g/kg/day. A preferred dosage is 0.05 g/kg/day to 20 g/kg/day, and a more preferred dosage is 0.1 g/kg/day to 5 g/kg/day. The administration may be administered once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, livestock, and humans by various routes. All modes of administration can be expected, and for example, it can be administered orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, intradural or intracerebrovascular injection, and skin application. The pharmaceutical composition of the present invention is a drug that can be safely used even for long-term administration for preventive purposes because it has almost no toxicity and side effects.

In another aspect, the present invention provides a food composition for preventing or improving a disease related to muscle loss, a food composition for improving muscle fatigue, and a food composition for improving muscle exercise ability, comprising an extract of Salvia miltiorrhizae, a fraction of the extract, or an effective ingredient derived from the extract or fraction. In a specific aspect, the food composition may be a health functional food.

The above food composition may contain a food-scientifically acceptable food additive.

The food composition may contain, but is not limited to, 0.001 to 90 wt%, 0.01 to 70 wt%, 0.1 to 50 wt%, or 1 to 30 wt% of the extract of Salvia militaris, a fraction of the extract, or an effective ingredient derived from the extract or isolate, based on the total weight of the composition.

The above food composition includes forms such as tablets, capsules, pills or liquids, and foods to which the extract of the present invention can be added include, for example, various foods, beverages, gum, tea, fermented milk containing lactic acid bacteria, vitamin complexes, health functional foods, etc.

In another aspect, the present invention provides a medium composition for muscle cell differentiation comprising a Dansam extract, a fraction of the extract, or an effective ingredient derived from the extract or fraction.

The medium composition according to the present invention may be used as a sole extract of Salvia militaris, but may include the extract of Salvia militaris in a basic medium used for cell culture, such as DMEM, and, if necessary, may further include a nutrient mixture, which is a mixture of various amino acids, vitamins, inorganic salts, etc., generally used in cell culture, as well as various organic and inorganic components acceptable for the culture medium depending on the intended use, such as ascorbic acid 2-glucoside (AA2G), insulin-transferrin-selenium, EGF, PDGF-BB, human serum, etc.

The concentration of the effective ingredient, the extract of Salvia militaris, the fraction of the extract, or the effective ingredient derived from the extract or fraction, included in the medium composition of the present invention may be included at a concentration required for differentiating myoblasts into muscle cells, and may be determined in consideration of the cell condition, required period, etc., and is not limited to a specific range of concentrations, but may be included in the medium composition at 0.01 to 99 wt%.

In another aspect, the present invention provides a method for muscle cell differentiation, comprising a step of culturing isolated myoblasts with a Salvia miltiorrhiza extract, a fraction of the extract, or an effective ingredient derived from the extract or fraction.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to illustrate the present invention, and the content of the present invention is not limited to the following examples.

[ Example ]

Example 1. According to the present invention Dansam Preparation of alcohol extract

The Salvia militaris extract according to the present invention was prepared as follows. Specifically, the Salvia militaris extract was prepared by extracting crushed Salvia militaris roots with ethanol (EtOH) at 50 to 80°C. (When extracting, the extraction can be performed using a 30 to 99 (v/v)% ethanol aqueous solution.)

The above-mentioned alcohol was extracted for 2 to 24 hours, and if necessary, it was extracted again for 2 to 12 hours in new alcohol, and then it was further extracted for 2 to 5 hours in new alcohol, and centrifuged to obtain a supernatant. Thereafter, the obtained supernatant was collected, and the extraction solvent was evaporated at 45 to 75°C, dried, and then ground and filtered through 80 mesh or larger to prepare.

Example 2. According to the present invention Dansam Preparation of water extract

A water extract of Dansam was prepared as in Example 1 above, using water instead of ethanol (EtOH).

Example 3. Alfalfa of the compressed material manufacturing

Alfalfa extract is obtained by collecting fresh alfalfa leaves, pressing them, removing the dry matter using a sieve, and obtaining the residue. A small amount of sodium carbonate is added to the obtained residue, and weak heat is applied using steam to precipitate the protein, and the precipitate is dried and made into a powder. As a result of analyzing the properties and content of the alfalfa extract, it was confirmed that the alfalfa extract was obtained as a green powder, and was composed of 54% protein, 11% fat, 10% mineral, and 13% fiber in weight %.

[ Experimental example ]

Experimental example 1. Example 1 of Dansam Analysis of the properties and contents of the extract

As a result of analyzing the properties and content of the Salvia militaris extract according to Example 1, it was confirmed that the Salvia militaris extract was obtained as a fine reddish brown powder and contained 10.3 wt% of cryptotanshinone, 8.8 wt% of tanshinone IIa, and 2.5 wt% of tanshinone I.

Experimental example 2. Example 1 of Dansam When taking the extract, it affects muscle tissue. AMPK Measurement of protein expression

To investigate the change in AMPK protein expression caused by the extract of Dansam in Example 1, C57BL/6J ^Lep For ob/ob mice, 0.47% (equivalent to 400 mg/kg, MB-1 group) and 0.7% (equivalent to 600 mg/kg, MB-2 group) of Salvia extract were mixed into the feed, pelleted, and allowed to ingest freely. The test substances were mixed and reprocessed, and the contents were analyzed and supplied as feed for 2 weeks. To compare the expression levels of AMPK, muscle tissues were extracted from samples of each group, washed with ice-cold PBS, ice-cold lysis buffer (1% Triton X-100) was added, and the tissues were incubated on ice for 30 minutes and centrifuged at 13,000 rpm for 15 minutes. The supernatant was separated to obtain intracellular proteins, and the protein concentration was measured using the Bradford method. 30 ug of protein was electrophoresed on a 10% SDS-polyacrylamide gel. The proteins separated by electrophoresis were transferred to nitrocellulose membranes and blocked with 5% skim milk for 2 hours at room temperature. The phspho-AMPK primary antibody was incubated at 4℃ for 16 hours. After washing with washing buffer (TBST with 0.1% Tween20), the secondary antibody was added and incubated at room temperature for 2 hours. As a result of confirming protein expression using the ECL system, it was confirmed that AMPK protein phosphorylation increased when treated with the Dansam extract. (See Figure 1)

Experimental example 3. Example 1 of Dansam Extract of In rats Pharmacokinetic studies for

In order to confirm the pharmacodynamic behavior of the Salvia militaris extract according to the present invention, a test was conducted using the Salvia militaris extract of Example 1 and a Salvia water extract as a comparative group, as follows.

Before the test, Spraque-Dawly rats (male, Samtaco Bio Korea Co., Ltd.) were fasted overnight before administration of the test substance, and were allowed free access to water. On the day of the test, 24 male rats, approximately 8 weeks old, were randomly assigned to 3 groups of 8 rats each and administered the Salvia militaris extract as shown in Table 1 below. At this time, the administration amount was calculated based on the body weight measured immediately before administration of the test substance, and each administration amount was orally administered using a sonde. Blood was collected from the jugular vein at 5, 10, 15, 20, 30, 40, 60, 120, 300, and 600 minutes after administration. The collected blood was immediately placed in a heparinized tube and centrifuged (12,000 rpm, 4℃, 2 minutes). After centrifugation, the separated plasma was placed in a 1.5 mL tube with label information and stored frozen at -70℃ or below until analysis. No clinical abnormalities related to the administration of the test substance were observed in any animal during the test period, and cryptotanshinone in the plasma was analyzed using LC-MS/MS, and the results are shown in Tables 1 and 2. At this time, the used Dansam ethanol extract A has a D90 of 109 μm, and the used Dansam ethanol extract G has a D90 of 18 μm.

Experimental example 4: Example 1 of Dansam extract, Nicotinamide mononucleotide (NMN), and/or alfalfa of the compressed material By combination Sarcopenia Check the improvement effect

To confirm the sarcopenia-improving effect of a combination of Salvia miltiorrhiza extract, nicotinamide mononucleotide (NMN), and alfalfa extract, the following test was conducted using a dexamethasone-induced muscle atrophy model (C2C12 myotube).

-Experimental model: Dexamethasone-induced muscle atrophy model (C2C12 myotube)

-Model derivation: When C2C12 myoblasts grew to about 90% of the well, they were replaced with 2% horse serum (HS). The 2% HS was replaced every two days. After culturing for 4 to 6 days, differentiation into myotubes was confirmed. Dexamethasone (10 or 100 μM) and the test substance were treated to myotubes for 24 or 48 hours.

-Experimental group:

(1) Control group

(2) Dexamethasone only (DEX) group

(3) DEX + 1 μg/ml group of the extract of Dansam of Example 1

(4) DEX + 5 μg/ml group of the extract of Dansam of Example 1

(5) DEX + 10μg/ml alfalfa extract group of Example 3

(6) DEX + 50μg/ml alfalfa extract group of Example 3

(7) DEX + 1 μg/ml of the extract of Dansam of Example 1 +

10μg/ml alfalfa extract group of Example 3

(8) DEX + 5 μg/ml of the extract of Dansam of Example 1 +

10μg/ml alfalfa extract group of Example 3

(9) DEX + 1 μg/ml of the extract of Dansam of Example 1 +

50μg/ml Alfalfa extract group of Example 3

(10) DEX + 5 μg/ml of the extract of Dansam of Example 1 +

50μg/ml Alfalfa extract group of Example 3

(11) DEX + 1 μg/ml of the hot water extract of Dansam of Example 2

(12) DEX + 5 μg/ml of the hot water extract of Dansam of Example 2

(13) DEX + 5 μg/ml of the extract of Dansam of Example 1 +

50μg/ml nicotinamide mononucleotide (NMN) group

(14) DEX + 5 μg/ml of the extract of Dansam of Example 1 +

5 μg/ml nicotinamide mononucleotide (NMN) group

(15) DEX + 50 μg/ml of the extract of Dansam of Example 1 +

5 μg/ml nicotinamide mononucleotide (NMN) group

(16) DEX + 5 μg/ml of the extract of Dansam of Example 1 +

50μg/ml Example 2 Dansam hot water extract group

(17) DEX + 5 μg/ml of the extract of Dansam of Example 1 +

5 μg/ml Example 2 Dansam hot water extract group

(18) DEX + 50μg/ml of the extract of Dansam of Example 1 +

5 μg/ml Example 2 Dansam hot water extract group

- Evaluation contents

(1) Myotube diameter

(2) NAD ⁺ & NADH level

(3) Protein expression

- Myosin heavy chain (MHC)

- SIRT1, PGC-1α

- p-AMPK & total AMPK

(4) Mitochondrial function

- Mitochondrial mass (by Mitotracker)

- Mitochondrial ROS (by MitoSox)

- ATP level

As a result of the experiment, it was confirmed that the Salvia militaris extract of Example 1 and the alfalfa extract of Example 2 had excellent sarcopenia-improving effects in the dexamethasone-induced muscle atrophy model (C2C12 myotube), and a synergistic sarcopenia-improving effect was confirmed when the Salvia militaris extract and the alfalfa extract were used together.

Experimental example 5. Cryptotanshinone , Tanshinone 2a , Salvianolic acid Sarcopenia Check the improvement effect

Using the same experimental method as Experimental Example 2, the sarcopenia-improving effects of cryptotanshinone, tanshinone 2a, salvianolic acid, and nicotinamide mononucleotide (NMN) were confirmed.

Experimental example 6. Dansam extract, Nicotinamide mononucleotide ( NMN ), and Alfalfa extract Including NAD ⁺ Promotional composite composition

The optimal mixing ratio that significantly and synergistically increases the sarcopenia improvement effect was confirmed by mixing various combinations of the Salvia extract of Example 1, the alfalfa extract of Example 3, and nicotinamide mononucleotide (NMN).

Claims (21)

A pharmaceutical composition for preventing or treating a disease related to muscle loss, characterized in that it contains a red sage extract as an active ingredient. In the first paragraph,
The above-mentioned Salvia militaris extract is a pharmaceutical composition for preventing or treating a disease related to muscle loss, characterized in that Salvia militaris is extracted with water, C1-C4 alcohol, or an alcohol aqueous solution thereof. In the second paragraph,
A pharmaceutical composition for preventing or treating a disease related to muscle loss, characterized in that the above-mentioned Dansam extract is extracted with water or a 50 to 100% (v/v) ethanol aqueous solution. In the third paragraph,
A pharmaceutical composition for preventing or treating a disease related to muscle loss, characterized in that the above-mentioned Dansam extract is a Dansam alcohol extract, a hot water extract, or a mixture thereof. In the first paragraph,
A pharmaceutical composition for preventing or treating a disease related to muscle loss, characterized in that the disease related to muscle loss is at least one selected from the group consisting of atony, muscular atrophy, sarcopenia, myasthenia, atonia, cachexia, and muscular dystrophy. In the first paragraph,
A pharmaceutical composition for preventing or treating a disease related to muscle loss, wherein the disease related to muscle loss is characterized by aging, cancer, kidney disease or diabetes. A pharmaceutical composition for preventing, improving or treating a disease related to muscle loss, characterized in that in claim 4, the Salvia militaris extract is a mixture of an alcohol extract and a hot water extract of Salvia militaris, and the weight ratio of the alcohol extract: hot water extract is 1:10 to 10:1. In the first paragraph,
A pharmaceutical composition for preventing or treating a disease related to muscle loss, characterized in that the above-mentioned ginseng extract contains at least one of tanshinone 2a, cryptotanshinone, and salvianolic acid B as an effective ingredient. In Article 8,
A pharmaceutical composition for preventing or treating a disease related to muscle loss, characterized in that the above-mentioned Dansam extract contains tanshinone 2a:cryptotanshinone in a weight ratio of 1:1 to 1:5. In Article 8,
A pharmaceutical composition for preventing or treating a disease related to muscle loss, characterized in that the above-mentioned Dansam extract contains tanshinone 2a:salvianolic acid in a weight ratio of 1:20 to 20:1. A pharmaceutical composition for preventing or treating a disease related to muscle loss, characterized in that the extract of Dansam of claims 1 to 4 further comprises an alfalfa extract. In Article 11,
A pharmaceutical composition for preventing or treating a disease related to muscle loss, characterized in that the above alfalfa extract is a filtrate obtained by pressing alfalfa leaves. In Article 11,
A pharmaceutical composition for preventing or treating a disease related to muscle loss, characterized in that the above-mentioned ginseng extract and alfalfa extract are contained in a weight ratio of ginseng extract:alfalfa extract of 1:100 to 1:1. A food composition for improving diseases related to muscle loss, characterized in that it contains as an effective ingredient an extract extracted from the water of Dansam, C1-C4 alcohol, or an alcohol aqueous solution thereof. A food composition for muscle growth characterized by containing as an effective ingredient an extract of Dansam using water, C1-C4 alcohol, or an alcohol aqueous solution thereof. A food composition for improving muscle fatigue, characterized in that it contains as an effective ingredient an extract of Dansam using water, C1-C4 alcohol, or an alcohol aqueous solution thereof. A food composition for improving muscle exercise ability, characterized in that it contains as an effective ingredient an extract of Dansam using water, C1-C4 alcohol, or an alcohol aqueous solution thereof. In any one of Articles 14 to 17,
A food composition characterized in that the above-mentioned Dansam extract further comprises nicotinamide mononucleotide (NMN). In Article 18,
The above food composition is a food composition characterized in that the weight ratio of the dansin extract:alfalfa extract is 1:1 to 1:100. A medium composition for muscle cell differentiation, characterized in that it contains as a derived component an extract extracted from the water of Dansam, C1-C4 alcohol, or an alcohol aqueous solution thereof. A composition for muscle cell differentiation, characterized in that the extract obtained by extracting water, C1-C4 alcohol, or an alcohol aqueous solution thereof from Dansam is cultured together with separated myoblasts.