COMPOSITION FOR PROMOTING MYOGENESIS, CONTAINING PROCESSED GINSENG EXTRACT

Abstract

The present invention relates to a composition for promoting myogenesis, containing, as an active ingredient, a processed ginseng extract in which a trace amount of a ginsenoside ingredient is increased. It has been ascertained that the processed ginseng extract promotes the differentiation of myoblasts into muscle and inhibits muscle atrophy caused by myostatin, which is a myogenesis inhibitory factor, and thus it is expected that a composition for preventing or treating muscle disorder-related diseases, having excellent effects, can be developed.

Claims

1. A composition for myogenesis promotion comprising a processed ginseng extract as an active ingredient.

2. The composition of claim 1, wherein the processed ginseng extract is prepared by: (a) seeding Aspergillus niger strain on a medium containing a ginseng powder and bran; (b) culturing the strain in step (a); (c) purifying the culture in step (b) through an ultrafiltration membrane; (d) separating an enzyme from the purification product in step (c); (e) adding the enzyme in step (d) to a ginseng powder, a red ginseng powder, a ginseng extract, or a red ginseng extract; (f) fermenting the product after addition in step (e); (g) separating the fermentation product in step (f); (h) concentrating a supernatant in step (g); (i) reacting the concentration product in step (h) with at least one organic acid selected from the group consisting of acetic acid, lactic acid, citric acid, malic acid, and tartaric acid; and (j) neutralizing, filtering, purifying, concentrating, and drying the reaction product in step (i).

3. The composition of claim 1, wherein the composition contains ginsenosides Rh2 and Rg3 each in 0.5-30 wt %.

4. The composition of claim 1, wherein the composition promotes myotube differentiation.

5. The composition of claim 1, wherein the composition inhibits myoatrophy caused by myostatin.

6. A pharmaceutical composition for prevention or treatment of a muscle disease, the pharmaceutical composition comprising the composition for myogenesis promotion of claim 1 and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition of claim 6, wherein the muscle disease is selected from the group consisting of muscular atrophy, myopathy, muscular injury, muscular dystrophy, myasthenia, sarcopenia, myoneural conductive disease, dermatomyositis, diabetic amyotrophy, nerve injury, amyotrophic lateral sclerosis (ALS), cachexia, and degenerative muscle diseases.

8. The pharmaceutical composition of claim 7, wherein the cachexia is caused by acquired immune deficiency syndrome (AIDS), celiac disease, multiple sclerosis, rheumatoid arthritis, chronic heart failure, congestive heart failure, uremia, tuberculosis, Crohn's disease, untreated or severe type 1 diabetes, anorexia nervosa, and hormone deficiency.

9. A health functional food for alleviation of a muscle disease, the health functional food comprising the composition for myogenesis promotion of claim 1 and a sitologically acceptable food supplement additive.

10. The health functional food of claim 9, wherein the muscle disease is selected from the group consisting of muscular atrophy, myopathy, muscular injury, muscular dystrophy, myasthenia, sarcopenia, myoneural conductive disease, dermatomyositis, diabetic amyotrophy, nerve injury, amyotrophic lateral sclerosis (ALS), cachexia, and degenerative muscle diseases.

11. The health functional food of claim 10, wherein the cachexia is caused by acquired immune deficiency syndrome (AIDS), celiac disease, multiple sclerosis, rheumatoid arthritis, chronic heart failure, congestive heart failure, uremia, tuberculosis, Crohn's disease, untreated or severe type 1 diabetes, anorexia nervosa, and hormone deficiency.

12. A method for treating a muscle disease in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a pharmaceutical composition comprising the composition for myogenesis promotion of claim 1 and a pharmaceutically acceptable carrier.

13. The method according to claim 12, wherein the muscle disease is selected from the group consisting of muscular atrophy, myopathy, muscular injury, muscular dystrophy, myasthenia, sarcopenia, myoneural conductive disease, dermatomyositis, diabetic amyotrophy, nerve injury, amyotrophic lateral sclerosis (ALS), cachexia, and degenerative muscle diseases.

14. The method of claim 12, wherein the cachexia is caused by acquired immune deficiency syndrome (AIDS), celiac disease, multiple sclerosis, rheumatoid arthritis, chronic heart failure, congestive heart failure, uremia, tuberculosis, Crohn's disease, untreated or severe type 1 diabetes, anorexia nervosa, and hormone deficiency.

15. The composition of claim 2, wherein the composition contains ginsenosides Rh2 and Rg3 each in 0.5-30 wt %.

16. A pharmaceutical composition for prevention or treatment of a muscle disease, comprising the composition for myogenesis promotion of claim 2 and a pharmaceutically acceptable carrier.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG. 1 shows the results of verifying whether the myoblast C2C12 cells differentiate into myotubes.

[0061] FIG. 2 shows the results of verifying cytotoxicity of a processed ginseng extract of the present invention.

[0062] FIG. 3 shows the results of verifying the myotube differentiation effect according to the treatment concentration and time of the processed ginseng extract of the present invention, wherein (A) shows cell images of myotubes; (B) shows the number of differentiated myotubes; and (C) shows the number of nuclei of differentiated myotubes.

[0063] FIG. 4 shows the results of verifying an increase in myotube breath according to the treatment concentration of the processed ginseng extract of the present invention, wherein (A) shows cell images of myotubes and (B) shows the measurement and digitalization results of myotube breadth.

[0064] FIG. 5 shows the results of verifying the degree of myotube atrophy according to the treatment concentration of myostatin (MSTN).

[0065] FIG. 6 shows the results of verifying an effect of inhibiting myotube atrophy caused by myostatin through the processed ginseng extract of the present invention, wherein (A) shows cell images of myotubes and (B) shows the measurement and quantification results of myotube breadth.

[0066] FIG. 7 shows the results of verifying the effect of inhibiting phosphorylation of Smad2, which is a signaling factor associated with myotube atrophy caused by myostatin, in the processed ginseng extract of the present invention, wherein (A) shows expression bands of phosphorylated Smad2 (pSmad2) and (B) shows the results of quantifying density of expression bands.

[0067] FIG. 8 shows the results of verifying the expression regulatory effects of MoyD, a core transcription factor in the expression of myofibrillar proteins, and MAFbx, an enzyme for degrading MyoD, in the processed ginseng extract of the present invention, wherein (A) shows the bands of confirming the protein expression of MyoD, MAFbx, and GAPDH; (B) shows the results of quantifying the expression of MAFbx compared with GAPDH in the presence or absence of the processed ginseng extract of the present invention; and (C) shows the results of quantifying the expression of MyoD compared with GAPDH in the presence or absence of the processed ginseng extract of the present invention.

MODE FOR CARRYING OUT THE INVENTION

[0068] Hereinafter, preferable examples of the present invention will be described in detail. However, the present invention is not limited to the examples described herein and can be embodied in many different forms. Rather, these examples are provided so that the present disclosure will be thorough and complete and will fully convey the scope of the disclosure to those skilled in the art.

[0069] The processed ginseng extracts of the present invention were manufactured by the methods disclosed in Korean Patent Registration Nos. 0992800 and 1595426.

Comparative Example 1: Preparation of Ginseng Powder

[0070] 200 g of 6-year-old ginseng was dried under hot air and then pulverized, to give 60 g of a ginseng powder.

Comparative Example 2: Preparation of Ginseng Concentrate

[0071] After 200 g of 6-year-old ginseng was dried under hot air, 1 L of 70% ethanol was added thereto, and then the mixture was stirred at 70 C. for 8 hours, followed by extraction, filtration, and concentration, to give 50 g of a ginseng concentrate.

Comparative Example 3: Preparation of Ginseng Concentrate Powder

[0072] After 200 g of 6-year-old ginseng was dried under hot air, 1 L of 70% ethanol was added thereto, and then the mixture was stirred at 70 C. for 8 hours, followed by extraction, filtration, concentration, and drying, to give 30 g of a ginseng concentrate powder.

Comparative Example 4: Preparation of Red Ginseng Powder

[0073] 200 g of 6-year-old ginseng was steamed at 98 C. for 1 hour and then dried, followed by pulverization, to give 40 g of a red ginseng powder.

Comparative Example 5: Preparation of Red Ginseng Concentrate

[0074] After 200 g of 6-year-old ginseng was steamed at 98 C. for 1 hour, 1 L of 70% ethanol was added thereto, and the mixture was stirred at 70 C. for 8 hours, followed by extraction, filtration, and concentration, to give 30 g of a ginseng concentrate.

Comparative Example 6: Preparation of Red Ginseng Concentrate Powder

[0075] After 200 g of 6-year-old ginseng was steamed at 98 C. for 1 hour, 1 L of 70% ethanol was added thereto, and the mixture was stirred at 70 C. for 8 hours, followed by extraction, filtration, concentration, and drying, to give 25 g of a red ginseng concentrate powder.

Comparative Example 7: Preparation of Ginseng Powder+0.2% Rh2+0.3% Rg3

[0076] 0.2 g of ginsenoside Rh2 and 0.3 g of ginsenoside Rg3 were mixed with 99.5 g of the ginseng powder of Comparative Example 1.

Comparative Example 8: Preparation of Red Ginseng Powder+0.2% Rh2+0.3% Rg3

[0077] 0.2 g of ginsenoside Rh2 and 0.3 g of ginsenoside Rg3 were mixed with 99.5 g of the red ginseng powder of Comparative Example 4.

Comparative Example 9: Preparation of Red Ginseng Powder+1% Rh2

[0078] 1 g of ginsenoside Rh2 was mixed with 99 g of the red ginseng powder of Comparative Example 4.

Comparative Example 10: Preparation of Red Ginseng Powder+1% Rg3

[0079] 1 g of ginsenoside Rg3 was mixed with 99 g of the red ginseng powder of Comparative Example 4.

Comparative Example 11: Preparation of Red Ginseng Powder+0.5% Rh2+0.5% Rg3

[0080] 0.5 g of ginsenoside Rh2 and 0.5 g of ginsenoside Rg3 were mixed with 99 g of the red ginseng powder of Comparative Example 4.

Example 1: Preparation of Processed Ginseng Powder Using Ginseng Powder

[0081] 250 g of a ginseng powder and 750 g of bran were added, and sterilized using a high-pressure steam sterilizer at 121 C. under 1.5 atm. The sterilized medium was mixed with 2 L of sterilized water, and then an Aspergillus niger suspension (510.sup.5 spores/g of medium weight) was seeded, and cultured at 28 C. for 7 days. Upon completion of culturing, a 0.02 M sodium acetate buffer was added and mixed, and then the resultant medium was filtered. The filtered culture was filtered using an ultrafiltration membrane (100 KDa or higher) and concentrated, to give 60 g of an enzyme liquid. 30 g of the enzyme liquid was added to 200 g of the ginseng powder of Comparative Example 1, followed by culturing at 28 C. for 18 hours, and then ethanol was added to precipitate an enzyme and the supernatant was concentrated. 2 L of purified water was added to 200 g of the concentrated product, and then 250 g of citric acid was added, followed by stirring at 50 C. for 18 hours. Upon completion of the reaction, 70% ethanol was added, followed by filtration and concentration, to give 200 g of a processed ginseng powder.

Example 2: Preparation of Processed Ginseng Concentrate Using Ginseng Concentrate

[0082] After 250 g of a ginseng powder and 750 g of bran were added, the mixture was sterilized using a high-pressure steam sterilizer at 121 C. under 1.5 atm. The sterilized medium was mixed with 2 L of sterilized water, and then an Aspergillus niger suspension (510.sup.5 spores/g of medium weight) was seeded, and cultured at 28 C. for 7 days. Upon completion of culturing, a 0.02 M sodium acetate buffer was added and mixed, and then the resultant medium was filtered. The filtered culture was filtered using an ultrafiltration membrane (100 KDa or higher) and concentrated, to give 60 g of an enzyme liquid. 30 g of the enzyme liquid was added to 200 g of the ginseng concentrate of Comparative Example 2, followed by culturing at 28 C. for 18 hours, and then ethanol was added to precipitate an enzyme and the supernatant was concentrated. After 2 L of purified water was added to 200 g of the concentrated product, 250 g of citric acid was added, followed by stirring at 50 C. for 18 hours. Upon completion of the reaction, 70% ethanol was added, followed by filtration and concentration, to give 190 g of a processed ginseng concentrate.

Example 3: Preparation of Processed Ginseng Concentrate Powder Using Ginseng Concentrate Powder

[0083] After 250 g of a ginseng powder and 750 g of bran were added, the mixture was sterilized using a high-pressure steam sterilizer at 121 C. under 1.5 atm. The sterilized medium was mixed with 2 L of sterilized water, and then an Aspergillus niger suspension (510.sup.5 spores/g of medium weight) was seeded, and cultured at 28 C. for 7 days. Upon completion of culturing, a 0.02 M sodium acetate buffer was added and mixed, and then the resultant medium was filtered. The filtered culture was filtered using an ultrafiltration membrane (100 KDa or higher) and concentrated, to give 60 g of an enzyme liquid. 30 g of the enzyme liquid was added to 200 g of the powdered ginseng concentrate of Comparative Example 3, followed by culturing at 28 C. for 18 hours, and then ethanol was added to precipitate an enzyme and the supernatant was concentrated. 2 L of purified water was added to 200 g of the concentrated product, and 250 g of acetic acid was added, followed by stirring at 50 C. for 8 hours. Upon completion of the reaction, 70% ethanol was added, followed by filtration, concentration, and drying, to give 195 g of a processed ginseng concentrate powder.

Example 4: Preparation of Processed Red Ginseng Powder Using Red Ginseng Powder

[0084] After 250 g of a ginseng powder and 750 g of bran were added, the mixture was sterilized using a high-pressure steam sterilizer at 121 C. under 1.5 atm. The sterilized medium was mixed with 2 L of sterilized water, and then an Aspergillus niger suspension (510.sup.5 spores/g of medium weight) was seeded, and cultured at 28 C. for 7 days. Upon completion of culturing, a 0.02 M sodium acetate buffer was added and mixed, and then the resultant medium was filtered. The filtered culture was filtered using an ultrafiltration membrane (100 KDa or higher) and concentrated, to give 60 g of an enzyme liquid. 30 g of the enzyme liquid was added to 200 g of the red ginseng powder of Comparative Example 4, followed by culturing at 28 C. for 18 hours, and then ethanol was added to precipitate an enzyme and the supernatant was concentrated. 2 L of purified water was added to 200 g of the concentrated product, and 250 g of acetic acid was added, followed by stirring at 50 C. for 8 hours. Upon completion of the reaction, 70% ethanol was added, followed by filtration, concentration, and drying, to give 195 g of a processed red ginseng powder.

Example 5: Preparation of Processed Red Ginseng Concentrate Using Red Ginseng Concentrate

[0085] After 250 g of a ginseng powder and 750 g of bran were added, the mixture was sterilized using a high-pressure steam sterilizer at 121 C. under 1.5 atm. The sterilized medium was mixed with 2 L of sterilized water, and then an Aspergillus niger suspension (510.sup.5 spores/g of medium weight) was seeded, and cultured at 28 C. for 7 days. Upon completion of culturing, a 0.02 M sodium acetate buffer was added and mixed, and then the resultant medium was filtered. The filtered culture was filtered using an ultrafiltration membrane (100 KDa or higher) and concentrated, to give 60 g of an enzyme liquid. 30 g of the enzyme liquid was added to 200 g of the red ginseng concentrate of Comparative Example 5, followed by culturing at 28 C. for 18 hours, and then ethanol was added to precipitate an enzyme and the supernatant was concentrated. 2 L of purified water was added to 200 g of the concentrated product, and 250 g of acetic acid was added, followed by stirring at 50 C. for 18 hours. Upon completion of the reaction, 70% ethanol was added, followed by filtration and concentration, to give 190 g of a processed red ginseng concentrate.

Example 6: Preparation of Processed Red Ginseng Concentrate Powder Using Red Ginseng Concentrate Powder

[0086] After 250 g of a ginseng powder and 750 g of bran were added, the mixture was sterilized by a high-pressure steam sterilizer at 121 C. under 1.5 atm. The sterilized medium was mixed with 2 L of sterilized water, and then an Aspergillus niger suspension (510.sup.5 spores/g of medium weight) was seeded, and cultured at 28 C. for 7 days. Upon completion of culturing, a 0.02 M sodium acetate buffer was added and mixed, and then the resultant medium was filtered. The filtered culture was filtered using an ultrafiltration membrane (100 KDa or higher) and concentrated, to give 60 g of an enzyme liquid. 30 g of the enzyme liquid was added to 200 g of the red ginseng concentrate of Comparative Example 6, followed by culturing at 28 C. for 18 hours, and then ethanol was added to precipitate an enzyme and the supernatant was concentrated. 2 L of purified water was added to 200 g of the concentrated product, and 250 g of acetic acid was added, followed by stirring at 50 C. for 8 hours. Upon completion of the reaction, 70% ethanol was added, followed by filtration, concentration, and drying, to give 195 g of a processed red ginseng powder.

[0087] Table 1 below shows the contents of ginsenosides Rh2 and Rg3 contained in the manufactured products of the examples and the comparative examples of the present invention through analysis by the method disclosed in Korean Patent Registration No. 992800. It was verified that the processed ginseng powders or processed ginseng extracts corresponding to Examples 1 to 6, of which trace ginsenosides are increased by producing a saponinase for a ginseng powder and a red ginseng powder and then using hydrolysis by the produced saponinase and an organic acid, contained large amounts of ginsenosides Rh2 and Rg3, which were increased compared with the ginseng powder and the red ginseng powder, which are target substances of reaction. Comparative Examples 7 to 11 were manufactured for comparison of a myogenesis promoting effect, wherein Comparative Examples 7 to 11 had the same contents of ginsenosides Rh2 and Rg3 as the examples of the present invention by, unlike the examples of the present invention, simply adding ginsenosides Rh2 and Rg3 to a ginseng powder and a red ginseng powder, which were not subjected to hydrolysis by a saponinase and an organic acid.

TABLE-US-00001 TABLE 1 Contents of ginsenosides Rg3 and Rh2 Content (wt %) Classification Rh2 Rg3 Example 1 (Processed ginseng powder) 0.2 0.3 Example 2 (Processed ginseng concentrate) 3 3 Example 3 (Processed ginseng concentrate powder) 12 18 Example 4 (Processed red ginseng powder) 0.6 0.8 Example 5 (Processed red ginseng concentrate) 1 3 Example 6 (Processed red ginseng concentrate 5 10 powder) Comparative Example 1 (ginseng powder) <0.01 <0.01 Comparative Example 2 (ginseng concentrate) <0.5 <0.5 Comparative Example 3 (ginseng concentrate powder) <0.5 <0.01 Comparative Example 4 (red ginseng powder) <0.01 <0.01 Comparative Example 5 (red ginseng concentrate) <0.5 <0.5 Comparative Example 6 (red ginseng concentrate <0.01 <0.01 powder) Comparative Example 7 (99.5 g of ginseng powder + 0.2 0.3 0.2 g of Rh2 + 0.3 g of Rg3) Comparative Example 8 (99.5 g of red ginseng powder + 0.2 0.3 0.2 g of Rh2 + 0.3 g of Rg3) Comparative Example 9 (99 g of red ginseng powder + 1 <0.01 1 g of Rh2) Comparative Example 10 (99 g of red ginseng powder + <0.01 1 1 g of Rg3) Comparative Example 11 (99 g of red ginseng powder + 0.5 0.5 0.5 g of Rh2 + 0.5 g of Rg3)

Test Example 1: Animal Cell Culture and Myotube Differentiation Induction

[0088] During myogenesis, myoblasts divided and fused to develop into myotubes, finally forming a muscle. Therefore, in order to investigate the effect of the processed ginseng extract of the present invention on myogenesis, the activity to differentiate into myotubes was investigated.

[0089] The mouse myoblast C2C12 (C2C12 ATCC CRL-1772) cells were used to investigate the activity to differentiate into myotubes. The C2C12 cells were cultured in a CO.sub.2 incubator with 5% CO.sub.2 at 37 C., in which Dulbecco's modified eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin were added.

[0090] The cultured C2C12 cells were dispensed into a plate, and grown until the cells reached a confluency of approximately 80-90%. The culture was removed, and DMEM containing 2% horse serum (Gibco) was added, followed by culturing, thereby inducing the differentiation into myotubes. Then, the formed myotubes were confirmed through a microscope, and the results are shown in FIG. 1.

[0091] As shown in FIG. 1, several myoblasts were fused to form tube-shaped myotubes after the induction of differentiation (B), unlike before differentiation (A).

[0092] It could be therefore seen that the treatment of C2C12 cells with horse serum could induce myogenesis.

Test Example 2: Verification of Cytotoxicity of Processed Ginseng Extracts

[0093] To investigate whether the cytotoxicity of the processed ginseng extract of the present invention, the myotubes obtained from differentiation in Test Example 1 were used.

[0094] The myotubes in Test Example 1 were dispensed in a 96-well plate, cultured for 24 hours, treated with the processed ginseng extract of the present invention at 0-100 g/ml, and then again cultured for 24 hours. After culturing, the intracellular ATP concentrations were measured using Promega's CellTiter-Glo Luminescent cell viability assay kit and a manual provided by the manufacturer, thereby determining cell viability. The results are shown in FIG. 2.

[0095] As shown in FIG. 2, regarding the processed ginseng extract of Example 2 among the processed ginseng extracts, the cell viability was slightly reduced up to a concentration of 30 g/ml, but reduced by about 7-12% at 50 g/ml and 100 g/ml compared with normal cells not treated with a processed ginseng extract. That is, it could be predicted that cytotoxicity may be shown when the concentration of the processed ginseng extract of Example 2 was 50 g/ml or higher.

[0096] Similar results could be obtained even in cases of the treatment with the processed ginseng extracts of the other examples.

[0097] It could be seen from the above results that the maximum treatment concentration of the processed ginseng extract of the present invention was 30 g/10.

Test Example 3: Verification of Myotube Formation Promotion by Processed Ginseng Extracts

[0098] During myogenesis, myoblasts divided and fused to develop into myotubes, finally forming a muscle. Therefore, to investigate the effect of the processed ginseng extract of the present invention on myogenesis, the activity to induce myotube formation was investigated.

[0099] A test was conducted by the same method as in the myotube formation induction test in Test Example 1. After the treatment with horse serum, the cells were treated with the processed ginseng extract of Example 2 at 0, 10, and 30 g/ml, and then cultured for 6 days. On days 0, 2, 4, and 6 of culture, the corresponding cells were fixed, and cell nuclei and cytoplasm were stained by Diff Quick kit (Sysmax, ZS0003, Japan) according to a manual provided by the manufacturer. Then, images were taken using a microscope, and the number of myotubes and the number of cell nuclei were analyzed, and the results are shown in FIG. 3.

[0100] As shown in FIG. 3, it was verified through cell images (A), the number of myotubes (B), and the number of cell nuclei (C) that myotubes were increased according to the treatment time and concentration in the treatment with the processed ginseng extract of Example 2.

[0101] In addition, the cells were treated with the processed ginseng extracts of Examples 1 to 6 and Comparative Examples 1 to 11 at 10 g/ml by the same method as in the above test, followed by culturing for 4 days, and then the number of myotubes was analyzed. The results are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Number of myotubes Classification per field Control (treatment with only horse serum) 25 Example 1 (Processed ginseng powder) 31 Example 2 (Processed ginseng concentrate) 37 Example 3 (Processed ginseng concentrate powder) 41 Example 4 (Processed red ginseng powder) 33 Example 5 (Processed red ginseng concentrate) 34 Example 6 (Processed red ginseng concentrate 40 powder) Comparative Example 1 (ginseng powder) 25 Comparative Example 2 (ginseng concentrate) 26 Comparative Example 3 (ginseng concentrate 27 powder) Comparative Example 4 (red ginseng powder) 26 Comparative Example 5 (red ginseng concentrate) 26 Comparative Example 6 (red ginseng concentrate 26 powder) Comparative Example 7 (99.5 g of ginseng powder + 30 0.2 g of Rh2 + 0.3 g of Rg3) Comparative Example 8 (99.5 g of red ginseng 31 powder + 0.2 g of Rh2 + 0.3 g of Rg3) Comparative Example 9 (99 g of red ginseng powder + 29 1 g of Rh2) Comparative Example 10 (99 g of red ginseng 29 powder + 1 g of Rg3) Comparative Example 11 (99 g of red ginseng 32 powder + 0.5 g of Rh2 + 0.5 g of Rg3)

[0102] As can be seen through Table 2 above, the numbers of myotubes in the treatment with the processed ginseng extracts of Comparative Examples 1 to 6 were similar to that in the control group treated with only horse serum, but the number of myotubes was increased in the treatment with the processed ginseng extracts of Examples 1 to 6. In addition, the number of myotubes was increased in the treatment with the processed ginseng extracts of Comparative Examples 7 to 11 in which ginsenosides Rh2 and Rg3 were added alone or together to the ginseng powder and the red ginseng powder of Comparative Examples 1 and 4 rather than the processed ginseng extracts of Comparative Examples 1 and 4, and especially, the number of myotubes was more in the treatment with the processed ginseng extracts of Comparative Examples 7, 8, and 11 in which ginsenosides Rh2 and Rg3 were added together rather than the treatment with the processed ginseng extracts of Comparative Examples 9 and 10 in which ginsenoside Rh2 or Rg3 each was added.

[0103] It can be seen from the above results that the co-administration of ginsenosides Rh2 and Rg3, like in the processed ginseng extracts of the present invention, increased a myogenesis promoting effect.

Test Example 4: Verification of Myotube Breadth Increasing Effect by Processed Ginseng Extracts

[0104] The myotubes formed during myogenesis are increased in length and breadth, and thus are fused with adjacent myotubes to form muscle fibers. Therefore, to investigate the effect of the processed ginseng extract of the present invention on myogenesis, the myotube breadth increasing effect was investigated.

[0105] After the C2C12 cells were differentiated into myotubes by the method of Test Example 1, the cells were treated with the processed ginseng extract of Example 2 at 0, 1, 10, and 30 g/0, followed by culturing for 24 hours. The cultured cells were fixed with 4% paraformaldehyde for 15 minutes, and treated with 0.2% triton X-100 for 15 minutes, and then blocked with 5% bovine serum albumin (BSA) for 6 hours. To observe cell morphology after blocking, an antibody against myosin heavy chain (MHC), which is a myotube forming and maintaining protein, were added, followed by incubation at 4 C. for 12 hours. Then, a secondary antibody conjugated to Alexa Fluor 488 fluorescent substance was incubated at room temperature for 4 hours, followed by immunofluorescent staining. Thereafter, the images of myotubes were observed using the fluorescent substance attached to MHC through a microscope, and the Image J program was used to determine the breadth of myotubes. The results are shown in FIG. 4. As for myotube breadth, the breadths of a total of 100 or more myotubes were measured at random in five regions, and quantified.

[0106] As can be seen from the images of myotubes in FIG. 4A and the measurement results of myotube breadth in FIG. 4B, the breadth of myotubes differentiated from C2C12 was increased according to the treatment concentration of the processed ginseng extract of Example 2.

[0107] Although not shown herein, it was verified that the treatment with the processed ginseng extracts of Examples 1, 3 to 5 also showed an increase in breadth of myotubes like the treatment with the processed ginseng extract of Example 2.

[0108] It can be seen from the above results that the processed ginseng extracts of the present invention had a myogenesis promoting effect by increasing the breadth of myotubes.

Test Example 5: Verification of Myoatrophy Inhibitory Effect of Processed Ginseng Extracts

Test Example 5-1: Construction of Myoatrophy Animal Cell Models

[0109] To investigate the myoatrophy inhibitory effect of the processed ginseng extracts of the present invention, myoatrophy animal cell models were constructed. The animal cell models were constructed on the basis of the fact that the breadth and length of myotubes are reduced in the presence of myoatrophy.

[0110] After myotubes were differentiated by the same method as in the induction of myotube differentiation of Test Example 1, the cells were added in DMEM not containing FBS, and cultured for 3 hours. After culturing, the cells were treated with the myogenesis inhibitor myostatin (MSTN) at different concentrations for 24 hours, and the change in breadth of myotubes was measured in order to investigate whether myoatrophy was induced. The change in breadth of myotubes was measured by the same method as in Test Example 4, and after the immunofluorescent staining, images of myotubes were observed using the fluorescent substance attached to MHC through a microscope, and the Image J program was used to determine the breadth of myotubes. The results are shown in FIG. 5.

[0111] As shown in FIG. 5, it was verified that the myotubes treated with myostatin, compared with the myotubes not treated with myostatin, were reduced in breadth of myotubes with the increase of the treatment concentration, and the myotubes treated with 0.4 g/0 myostatin resulted in a greatest reduction in breadth of myotubes.

[0112] Accordingly, the treatment with 0.4 g/0 myostatin was selected in the construction of myoatrophy animal cell models for investigating the myoatrophy inhibitory effect of the processed ginseng extracts of the present invention.

Test Example 5-2: Verification of Myoatrophy Inhibitory Effect of Processed Ginseng Extracts

[0113] To investigate the myoatrophy inhibitory effect of the processed ginseng extracts of the present invention, myoatrophy was induced by the same method as the construction method of myoatrophy animal cell models in Test Example 5-1, and then the change in myotube breadth was observed. The results are shown in FIG. 6. The myotubes were treated with 0.4 g/0 myostatin (MSTN) and 30 g/0 the processed ginseng extract of Example 2 alone or in combination, and the myotubes not treated with myostatin and the processed ginseng extract of Example 2 were used as a control group.

[0114] It can be seen from the images of myotubes in FIG. 6A and the measurement results of myotube breadth in FIG. 6B that the myotube breadth was increased in the treatment with the processed ginseng extract of Example 2 compared with the control group while the myotube breadth was reduced in the treatment with myostatin (MSTN). It was also verified that the myotube breadth reduced by myostatin was increased in the treatment with myostatin and the processed ginseng extract of Example 2 together.

[0115] It can be seen from the above results that the processed ginseng extracts of the present invention could increase the myotube breadth and inhibit myoatrophy caused by myostatin during myogenesis.

Test Example 6: Verification of Myostatin-Associated Signaling Inhibitory Effect of Processed Ginseng Extract

Test Example 6-1: Verification of Smad2 Phosphorylation Inhibitory Effect

[0116] Myostatin (MSTN) is known to induce myoatrophy by increasing ubiquitin proteasome pathway (UPP)-mediated protein degradation through activation of the activin type II receptor (ActRIIB)-Smad2 signaling pathway. Therefore, to investigate the inhibitory effect of the processed ginseng extracts of the present invention on myoatrophy caused by myostatin, it was examined whether or not Smad2 phosphorylation was inhibited in the ActRIIB-Smad2 pathway.

[0117] The myotubes differentiated by the same method as in Test Example 5-2 were treated with myostatin and the processed ginseng extract of Example 2 to secure cells, and the cells were subjected to Western blotting. The treatment concentrations of the processed ginseng extract of Example 2 was 0, 10, 30, and 100 g/ml. A lysis buffer was added to the secured cells to lyse cells, and then centrifugation was used to secure proteins of the supernatant. The secured proteins of the supernatant were separated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked by a blocking solution, treated with a primary antibody in order to determine the amounts of Smad2 and phosphorylated Smad2 (pSmad2), and then treated with a secondary antibody to the primary antibody. Then, enhanced chemiluminescence (ECL, Amersham) was used to examine protein expression. The protein band density was quantified, and the results are shown in FIG. 7.

[0118] As can be seen from the protein expression band results in FIG. 7A and the results of quantifying expression levels in FIG. 7B, it was verified that Smad2 phosphorylation (pSmad2) was increased when the myotubes were treated with myostatin (MSTN) while Smad2 phosphorylation (pSmad2) was reduced with the increase of the treatment concentration when the myotubes were treated with the processed ginseng extract of Example 2.

Test Example 6-2: Verification of MyoD Degradation Pathway Inhibitory Effect

[0119] It has been known that the degradation of MyoD, a core transcription factor in the expression of myofibrillar proteins important for myogenesis, is driven by the UPP procedure activated via the myostatin (MSTN)-ActRIIB-Smad2 pathway. The UPP procedure is regulated by ubiquitinated E3 ligases specialized to a protein of interest, and MAFbx is known to be the E3 ligase of MyoD, which is a core in this procedure. Therefore, the changes in MyoD and MAFbx contents were examined to investigate the inhibitory effect of the processed ginseng extract of the present invention on myoatrophy caused by myostatin.

[0120] The myotubes differentiated by the same method as in Test Example 5-2 were treated with myostatin and the processed ginseng extract of Example 2 to secure cells. The treatment concentrations of the processed ginseng extract of Example 2 was 30 g/ml. The secured cells were subjected to Western blotting using antibodies corresponding to MyoD and MAFbx by the same method as in Test Example 6-1, and the results are shown in FIG. 8.

[0121] As shown in the MAFbx and MyoD protein expression band results in FIG. 8A and the quantification results of MAFbx (FIG. 8B) and MyoD (FIG. 8C) expression levels, the treatment of myotubes with myostatin (MSTN) resulted in an increase in MAFbx expression and a reduction in MyoD expression, while the treatment with the processed ginseng extract of Example 2 resulted in a reduction in MAFbx expression increased by MSTN and an increase in MyoD expression reduced by MSTN.

[0122] It can be seen through the results of Test Examples 6-1 and 6-2 that the processed ginseng extracts of the present invention promoted myogenesis by inhibiting signaling of the myogenesis inhibitor myostatin.