A COMPOSITION COMPRISING AN EXTRACT OF ALDER TREE OR THE ISOLATED COMPOUNDS THEREFROM FOR TREATING AND PREVENTING SKELETON MUSCLE-RELATED DISORDER AND THE USE THEREOF

Abstract

The present invention related to a composition comprising an extract of alder tree or the isolated compounds therefrom for treating and preventing skeleton muscle disease and the use thereof. It has been confirmed that the inventive extract/compounds showed potent improving or treating effect on muscle loss through various in vitro test and animal model tests, for example, Stimulation effect of inventive extract/compounds on the initiation effect of myoblast differentiation (Experimental Example 1, 3 and 4), confirming that inventive extract/compounds increases the number of cylinder-shaped multinucleated myotubes, resulting in promoting muscle cell differentiation; Study on p38 MAPK signaling system mechanism of inventive extract/compounds for promoting myoblast differentiation (Experimental Example 2 and 5); Inhibitory effect of inventive extract/compounds on muscle loss (Experimental Example 6 and 7); improving effect on motility in animal model of muscle loss (In vivo) (Experimental Example 8) confirming that s that inventive extract/compounds promotes muscle cell differentiation as well as improves motor capacity decline and muscle loss. Therefore, the inventive extract/compounds of the present invention can be usefully used in a pharmaceutical composition, health functional food, and health supplement food for preventing or treating on skeleton muscle disease.

Claims

1. A pharmaceutical composition comprising an extract of alder tree or the compounds isolated therefrom selected from (−)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol (DFS, compound 1), platyphyllenone (compound 2), (5R)—O-methylhirsutanonol (compound 3), hirsutanonol (compound 4), platyphylloside (compound 5) or oregonin (compound 6) as an active ingredient for the treatment or prevention of skeletal muscle diseases.

2. The pharmaceutical composition according to claim 1, said alder tree is selected from Alnus japonica (Thunb.) Steudel or Alnus japonica var. koreana.

3. The pharmaceutical composition according to claim 1, said alder tree is extracted from the group consisting of the root part, stem part, bark part, xylem part, herb part and leaf part of alder tree.

4. The pharmaceutical composition according to claim 1, said extract of alder tree is selected from crude extract, non-polar solvent soluble extract or non-polar solvent soluble extract of alder tree.

5. The pharmaceutical composition according to claim 1, said crude extract of alder tree is dissolved in polar solvent selected from distilled water, spirit, methanol, ethanol, butanol and the like, or the mixtures thereof.

6. The pharmaceutical composition according to claim 1, said skeletal muscle disease is selected from the group consisting of muscular atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle stiffness, amyotrophic axonal sclerosis, myasthenia gravis, cachexia and sarcopenia.

7. (canceled)

8. (canceled)

9. A treating agent of cachexia or senile muscular atrophy comprising an extract of alder tree or the compounds isolated therefrom selected from (−)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol (DFS, compound 1), platyphyllenone (compound 2), (5R)—O-methylhirsutanonol (compound 3), hirsutanonol (compound 4), platyphylloside (compound 5) or oregonin (compound 6) as an active ingredient.

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. A health functional food comprising an extract of alder tree or the compounds isolated therefrom selected from (−)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol (DFS, compound 1}, platyphyllenone (compound 2), (5R)—O-methylhirsutanonol (compound 3), hirsutanonol (compound 4), platyphylloside (compound 5) or oregonin (compound 6) as an active ingredients for the improvement or prevention of skeletal muscle diseases.

16. The health functional food according to claim 15, said health food is provided as powder, granule, tablet, chewing tablet, capsule or beverage type.

17. (canceled)

18. (canceled)

19. A health supplement food comprising an extract of alder tree or the compounds isolated therefrom selected from (−)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol (DFS, compound 1}, platyphyllenone (compound 2), (5R)—O-methylhirsutanonol (compound 3), hirsutanonol (compound 4), platyphylloside (compound 5) or oregonin (compound 6) as an active ingredients for the improvement or prevention of skeletal muscle diseases.

20. (canceled)

21. (canceled)

22. A food additive comprising an extract of alder tree or the compounds isolated therefrom selected from (−)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol (DFS, compound 1}, platyphyllenone (compound 2), (5R)—O-methylhirsutanonol (compound 3), hirsutanonol (compound 4), platyphylloside (compound 5) or oregonin (compound 6) as an active ingredients for the improvement or prevention of skeletal muscle diseases.

23. (canceled)

24. (canceled)

25. A method of treating or preventing skeletal muscle diseases in a mammal suffering from skeletal muscle diseases comprising administering to said mammal an effective amount of an extract of alder tree or the compounds isolated therefrom selected from (−)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol (DFS, compound 1), platyphyllenone (compound 2), (5R)—O-methylhirsutanonol (compound 3), hirsutanonol (compound 4), platyphylloside (compound 5) or oregonin (compound 6).

26. (canceled)

Description

DESCRIPTION OF DRAWINGS

[0074] FIG. 1 depicts the stimulation effect of inventive extract on MHC and myoD expression;

[0075] FIG. 2 shows the effect of inventive extract on MHC expression in the number of cylinder-shaped multinucleated myotubes;

[0076] FIG. 3 presents the effect of inventive extract on p38 MAPK signaling system mechanism;

[0077] FIG. 4 represents the stimulation effect of inventive compounds 1-6 on MHC expression;

[0078] FIG. 5 depicts shows the effect of inventive compounds 1-6 on MHC expression in the number of cylinder-shaped multinucleated myotubes;

[0079] FIG. 6 shows the stimulation effect of inventive compound 1 on MHC and myoD expression;

[0080] FIG. 7 presents the effect of inventive compound 1 on MHC expression in the number of cylinder-shaped multinucleated myotubes;

[0081] FIG. 8 represents the effect of inventive compound 1 on p38 MAPK signaling system mechanism;

[0082] FIG. 9 depicts the effect of inventive extract on MHC and MAFbx expression in the myotube cell treated with dexamethasone to indue muscle loss;

[0083] FIG. 10 shows the stimulation effect of inventive extract on MHC expression of cylinder-shaped multinucleated myotubes in the myotube cell treated with dexamethasone to indue muscle loss;

[0084] FIG. 11 shows the stimulation effect of inventive compounds 1-6 on MHC expression of cylinder-shaped multinucleated myotubes in the myotube cell treated with dexamethasone to indue muscle loss.

BEST MODE

[0085] It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

[0086] The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

Examples

[0087] The following Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Example 1. Preparation of a Crude Extract of Alnus japonica Steud

[0088] 3 L of 100% ethanol (spirit) was added to 600 g of the stem including the bark and xylem of the dried Alnus japonica steud (Yeongcheon, North Gyeongsang Province, Korea) and extracted at room temperature for 12 hours to extracted solution. The solution was filtered and concentrated under reduced pressure conditions. The concentrated ethanol (spirit) extract was lyophilized in a freeze dryer (ScanVac, Labogene) under reduced pressure conditions to obtain 28 g of dried powder of crude extract of Alnus japonica steud (hereinafter referred to as AJE) and used in the next experiment.

Example 2. Preparation of a Purified Fractions of Alnus japonica Steud

[0089] 28 g of dried powder of crude extract of Alnus japonica steud obtained in Example 1 is suspended by adding 500 mL of purified water, 500 mL of n-hexane is added to the suspension to fractionate into n-hexane soluble fraction. 500 mL of chloroform, 500 mL of ethyl acetate and 500 mL of butanol are sequentially added to the remaining water suspension to afford each fraction. Each fraction was dried under reduced pressure respectively to afford 4.1 g of n-hexane soluble fraction (hereinafter referred to as AJH), 2.5 g of chloroform soluble fraction (hereinafter referred to as AJC), 2.9 g, and ethyl acetate soluble fraction (hereinafter referred to as AJE) and 1.8 g of butanol soluble fraction (hereinafter referred to as AJB), respectively.

Example 3. Isolation of Inventive Compounds from Ration of an Extract of Alnus japonica Steud

[0090] In order to find active ingredient from an extract of Alnus japonica steud obtained in the above, 2.5 g of chloroform soluble fraction (AJC) was further purified with silica gel column chromatography using mixture solvent system (n-hexane/acetone gradient system, 50:1.fwdarw.1:2) to afford 3 subfractions (CF1, CF2 and CF3) and the CF2 subfraction was further purified with reversed-phase column chromatography using mixture solvent system (MeOH gradient system, 50%.fwdarw.100%) to afford compound 1 ((−)-(2R,3R)-1,4-O-diferuloylsecoisorriscinol {(−)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol (DFS) compound 1, 23 mg).

[0091] 2.9 g of ethyl acetate soluble fraction was further purified with silica gel column chromatography using by chloroform/methanol gradient method to obtain 13 fractions (EF1˜EF13). The EF3 fraction was further purified with silica gel column chromatography using by hexane/ethyl acetate solvent conditions to obtain 7 subfractions (EF1-EF7) and the EF3-3 subfraction was further purified with silica gel column chromatography using hexane/acetone solvent conditions to afford compound 2 (10.9 mg).

[0092] The EF8 fraction was further purified with silica gel column chromatography using by dichloromethane/methanol solvent conditions to afford 3 subfractions (EF8-1˜3) and the EF8-2 subfraction was again purified with RP-MPLC under 10%-100% methanol solvent conditions to afford several subfractions. the EF8-2-2 among the subfractions was further purified with HPLC Prep. Using by 50% methanol solvent condition to obtain compound 3 (9.5 mg). The EF10 fraction was further purified with MPLC using by 10% to 100% acetonitrile solvent condition and then HPLC prep under 50% methanol to afford compound 4 (8.5 mg), compound 5 (15 mg) and compound 6 (8.6 mg).

[0093] Compound 1 was identified as (−)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol (DFS, a lignan derivative) with more than 95% purity by comparing the physicochemical data of HPLC and .sup.1H-NMR with those disclosed in previous literature (J. Chem. Soc. Chem. Commun., 1975, 9:316).

[0094] Compounds 2, 3, 4, 5, and 6 were identified as diarylheptanoid derivatives, platyphyllenone (compound 2: Chem. Pharm. Bull. 1996, 44:1033), (5R)—O-methylhirsutanonol (compound 3: Chem. Pharm. Bull. 2006, 54:139), hirsutanonol (compound 4: J. Nat. Prod., 1998, 61:1292), platyphylloside (compound 5: Phytochem. 1993, 32:365), oregonin (compound 6: Chem Pharm Bull., 2010, 58: 238), respectively, by comparing the physicochemical data of HPLC and .sup.1H-NMR with those disclosed in previous literatures.

##STR00001##

[0095] (A) Compound 1: (−)-(2R,3R)-1,4-O-diferuloylsecoisolariciresinol (DFS)

[0096] appearance: white powder

[0097] Molecular formulae: C.sub.40H.sub.42O.sub.12 (MW. 714.27);

[0098] .sup.1H-NMR (DMSO-d.sub.6, 400 MHz): δ9.59 (1H, brs, —OH), 8.69 (1H, brs, —OH), 7.52 (2H, d, J=15.8 Hz, H-7″, 7″′), 7.29 (2H, d, J=1.9 Hz, H-2″, 2″′), 7.09 (2H, dd, J=8.3, 1.9 Hz, H-6″, 6″′), 6.77 (2H, d, J=8.1 Hz, H-5″, 5″′), 6.66 (2H, s, J=1.4 Hz, H-2, 2′), 6.64 (2H, s, H-5, 5′), 6.53 (2H, dd, J=8.0, 1.8 Hz, H-6, 6′), 6.48 (1H, s, J=15.9 Hz, H-8″′), 6.46 (1H, d, J=15.9 Hz, H-8″), 4.26 (2H, dd, J=11.2, 6.4 Hz, H-9), 4.07 (2H, dd, J=11.3, 5.0 Hz, H-9′), 3.80 (3H, s, —OCH.sub.3), 3.67 (3H, s, —OCH.sub.3), 2.75 (2H, dd, J=13.6, 6.1 Hz, H-7), 2.55 (2H, dd, J=13.6 Hz, H-7′), 2.19 (2H, s, H-8, 8′) .sup.13C-NMR (DMSO-d.sub.6, 100 MHz): δ166.6 (C-9″, 9″′), 149.3 (C-4″, 4″′), 147.9 (C-3″, 3″′), 147.4 (C-3, 3′), 145.0 (C-7″, 7″′), 144.6 (C-4, 4′), 130.8 (C-1, 1′), 125.5 (C-1″, 1″′), 123.1 (C-6″, 6″′), 120.9 (C-6, 6′), 115.5 (C-5″, 5″′), 115.2 (C-5, 5′), 114.4 (C-8″, 8″′), 112.8 (C-2, 2′), 111.2 (C-2″, 2″′), 64.8 (C-9, 9′), 55.6 (—OCH.sub.3), 55.3 (—OCH.sub.3), 39.10 (C-8, 8′), 34.7 (C-7, 7′)

[0099] (B) Compound 2: Platyphyllenone

[0100] appearance: yellow oil

[0101] Molecular formulae: C.sub.19H.sub.20O.sub.3(MW. 296.14);

[0102] .sup.1H NMR (CD.sub.3OD, 500 MHz) δ: 6.98 (4H, m, aromatic protons), 6.87 (1H, d, J=15.9 Hz, H-5), 6.68 (4H, m, aromatic protons), 6.05 (1H, dt, J=15.9, 1.4 Hz, H-4), 2.78 (4H, m, H-1, 2), 2.66 (2H, t, J=7.5 Hz, H-6), 2.45 (2H, m, H-7).

[0103] .sup.13C NMR (CD.sub.3OD, 125 MHz) δ: 202.8 (C-3), 156.69 (C-4′), 156.64 (C-4″), 149.22 (C-5), 133.18 (C-1′), 132.99 (C-1″), 131.64 (C-4), 130.37 (C-2′, 2″), 130.31 (C-6′, 6″), 116.17 (C-3′, 3″), 116.15 (C-5′, 5″), 42.7 (C-2), 35.7 (C-7), 34.5 (C-6), 30.6 (C-1).

[0104] (C) Compound 3: (5R)—O-Methylhirsutanonol

[0105] appearance: brown oil

[0106] Molecular formulae: C.sub.20H.sub.24O.sub.6(MW. 360.16);

[0107] .sup.1H NMR (CD.sub.3OD, 500 MHz) δ: 6.67 (2H, dd, J=7.9, 4.8 Hz, aromatic proton), 6.62 (2H, d, J=2.9 Hz, aromatic protons), 6.50 (2H, dd, J=9.7, 3.8 Hz, aromatic protons), 3.65 (1H, dt, J=12.0, 6.0 Hz, H-5), 3.28 (3H, s, —OCH.sub.3), 2.68 (5H, m, H-1, 2), 2.50 (3H, m, H-7), 2.50 (1H, m, H-4), 1.71 (2H, m, H-6); .sup.13C NMR (CD.sub.3OD, 125 MHz) δ: 211.59 (C-3), 146.18 (C-4′), 146.15 (C-4″), 134.80 (C-1′), 134.02 (C-1″), 120.61, 120.56 (aromatic carbon), 116.51, 116.49, 116.34 (aromatic carbon), 77.93 (C—OCH.sub.3), 57.06 (C-5), 48.25 (C-4), 46.35 (C-1), 36.97 (C-6), 31.63 (C-7), 30.10 (C-2).

[0108] (D) Compound 4: Hirsutanonol

[0109] appearance: yellow oil

[0110] Molecular formulae: C.sub.19H.sub.22O.sub.6(MW. 346.14);

[0111] .sup.1H NMR (CD.sub.3OD, 500 MHz) δ: 6.65 (4H, m, aromatic proton), 6.51 (2H, ddd, J=8.0, 3.7, 2.0 Hz, aromatic protons), 4.02 (1H, dq, J=12.5, 6.3 Hz, H-3), 2.72 (4H, m, H-1, 2), 2.54 (4H, m, H-4, 7), 1.67 (2H, m, H-6); .sup.13C NMR (CD.sub.3OD, 125 MHz) δ: 212.03 (C-3), 146.17 (C-3′), 146.12 (C-3″), 144.46 (C-4′), 144.23 (C-4″), 134.94 (C-1′), 134.07 (C-1″), 120.64 (C-6′), 120.51 (C-6″), 116.54 (C-2′), 116.46 (C-5′), 116.34 (C-2″), 116.30 (C-5″), 68.31 (C-5), 51.26 (C-4), 46.37 (C-2), 40.44 (C-6), 32.17 (C-7) 30.04 (C-1).

[0112] (E) Compound 5: Platyphylloside

[0113] appearance: brown oil

[0114] Molecular formulae: C.sub.25H.sub.32O.sub.9(MW. 476.52);

[0115] .sup.1H NMR (CD.sub.3OD, 500 MHz) δ: 7.02 (1H, t, J 8.5 Hz, aromatic protons), 6.69 (1H, dd, J=8.4, 0.8 Hz, aromatic protons), 4.30 (1H, d, J 7.8 Hz, H-1″′), 4.18 (1H, m, H-5), 3.88 (1H, dd, J=11.8, 2.3 Hz, H-6″′b), 3.72 (1H, dd, J=11.8, 5.3 Hz, H-6″′a), 3.36 (2H, dd, J=16.5, 8.6 Hz, H-3″′, 4″′,), 3.26 (1H, m, H-5″′), 3.16 (1H, t, J=8.4 Hz, H-2″′), 2.82 (1H, dd, J=16.6, 6.8 Hz, H-4b), 2.77 (4H, d, J=8.1 Hz, H-1, 2), 2.60 (3H, m, H-4a, 7), 1.85 (1H, ddd, J=13.6, 8.6, 6.9 Hz, H-6b), 1.75 (1H, m, H-6a); .sup.13C NMR (CD.sub.3OD, 125 MHz) δ: 211.88 (C-3), 156.58 (C-4′), 156.30 (C-4″), 134.33 (C-1″), 133.23 (C-1′), 130.43 (C-2, 2″), 130.33 (C-6′, 6″), 116.18 (C-3′, 3″), 116.05 (C-5′, 5″), 103.46 (C-1″′), 78.05 (C-3″′), 77.83, (C-5″′), 76.21 (C-5), 75.21 (C-2″′), 71.59 (C-4″′), 62.75 (C-6″′), 48.69 (C-4), 46.41 (C-2), 38.51 (C-6), 31.43 (C-7), 29.82 (C-1).

[0116] (F) Compound 6: Oregonin

[0117] appearance: brown powder

[0118] Molecular formulae: C.sub.24H.sub.30O.sub.10 (MW 478.49)

[0119] .sup.1H NMR (Me.sub.2SO-d.sub.6+D.sub.2O, 500 MHz) δ: 6.71-6.75 (4H, H-2′, 2″, 5′, 5″), 6.52 (2H, dd, J=8.1, 2.1 Hz, H-6′, 6″), 4.32 (1H, d, J=7.8 Hz, xyl-1), 4.14 (1H, m, H-5), 3.90 (1H, dd, J=11.4, 6.1 Hz, xyl-5 eq), 3.56 (1H, m, xyl-4), 3.48 (1H, dd, J=9.0, 9.0 Hz, xyl-3), 3.23 (1H, dd, J=11.4, 11.2 Hz, xyl-5a), 3.21 (1H, dd, J=9.0, 7.8 Hz, xyl-2), 2.52-2.86 (8H, H-1, 4, 6, 7), 1.75-1.82 (2H, m, H-2). .sup.13C NMR (Me.sub.2SO-d.sub.6+D.sub.2O, 125 MHz) δ: 211.0 (C-3), 145.7 (C-3″), 145.3 (C-3′), 144.1 (C-4″), 143.8 (C-4′), 134.8 (C-1″), 133.8 (C-1′), 120.4 (C-6″), 120.4 (C-6′), 116.4 (C-5″), 116.4 (C-5′), 116.2 (C-2″), 116.1 (C-2′), 103.8 (xyl-1), 77.3 (xyl-3), 76.1 (C-5), 74.5 (xyl-2), 70.6 (xyl-4), 66.4 (xyl-5), 48.1 (C-4), 46.0 (C-2), 38.1 (C-6), 31.3 (C-7), 29.2 (C-1).

Experimental Example 1. Stimulation Effect of Inventive Extract on the Initiation Effect of Myoblast Differentiation

[0120] In order to investigate the myogenic effect of the inventive extract obtained in Examples on the myoblast differentiation, following experiment was performed according to the procedure disclosed in the previous literature (Chem. Biol. Interact. 2016, 248:60).

[0121] 1-1. Test Procedure

[0122] Various concentrations of inventive extract (1, 10, 100 ng/ml) were treated to C2C12 cells (mouse myoblast cell line; Ca.sup.#CRL-1771, American Type Culture Collection (ATCC)) and cell lysates obtained from the differentiated cells, were subjected to Western Blot analysis to evaluate the expressed level of MHC and myoD proteins (The result was shown in FIG. 1A). the change of MHC expression caused by inventive extract in myotube cell was determined by immunostaining method using by mouse anti-MHC and anti-mouse IgG2b Alexa-fluor 568 (See FIG. 1B) (Chem. Biol. Interact. 2016, 248:60).

[0123] Various concentrations of inventive extract (1, 10, 100 ng/ml) were treated to C2C12 cells (mouse myoblast cell line; Cat.sup.#CRL-1771, ATCC) in differentiation medium (2% horse serum-containing DMEM, Cat.sup.#11965-084, Gibco) for 3 days to induce their differentiation.

[0124] 1-1-1. Western Blot Analysis

[0125] For Western blot analysis, about 3×10.sup.4 mouse myoblast cell cells were incubated in 60 mm plates for 24 hours, and each sample was added to the differentiation medium for 3 days to differentiate. The protein extract was obtained from the differentiated cells using by lysis buffer [25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail, Calbiochem, Darmstadt, Germany). The protein extract (25 μg) was performed to SDS-polyacrylamide gel electorphoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes.

[0126] Primary mouse anti-MHC (sc-376157, Santa Cruz) or anti-myoD (sc-32758, Santa Cruz) was conjugated to the blots at 4° C., for 12 hrs and then conjugated with anti-mouse secondary antibody conjugated to Horseradish peroxidase (goat anti-mouse IgG-HRP, sc-2005, SantaCruz) to analyze the amount of MHC protein by chemiluminescence method. Pan-cadherin (Cat #3678, Sigma) was used as a loading control in the experiment.

[0127] 1-1-2. Immunofluorescence Staining

[0128] According to the above-described method, the differentiated myoblast cells in the differentiation medium were subject to immunofluorescence staining method.

[0129] After removing respective differentiation medium, the differentiated myoblast cells in the differentiation medium were washed with phosphate buffer solution twice and fixed with 4% paraformaldehyde (0141, BBC Biochemical) for 20 mins. After washing the cells with phosphate buffer solution twice again, the cells were treated with 0.1% tritonX-100 (2315025, Sigma) for 20 mins. After washing the cells with phosphate buffer solution twice again, the cells were blocked with 5% horse serum solution (16050122, Gibco) and mouse anti-MHC (MAB4470, R&D systems) was added thereto to react for 12 hours at 4° C.

[0130] After finishing the reaction, the cells were washed with phosphate buffer solution more than 3 times and then the level of MHC expression was analyzed using by Alexa Flouor 568-conjuagted secondary anti-mouse (A-21144, MicoProbes) and DAPI (D9542, Sigma). The test result of immunofluorescence on MHC-positive myotubes was visualized with red color and the DAPI-labelled nucleus was visualized with blue color.

[0131] 1-2. Test Result (See Table 1 and FIG. 1-2)

[0132] As shown in FIG. 1, the effect of test sample on the expression of MHC and myoD was determined. The cells added with various concentrations of inventive extract (1, 10, 100 ng/ml) were treated to myotubes differentiation medium to be differentiate to muscle fiber for 3 days. The muscle fibers were collected and the level of differentiation markers to muscle fibers, i.e., MHC and myoD protein was analyzed according to Western blot analysis.

[0133] At the result, it can be confirmed that the test sample group treated with inventive extract showed increased expression level comparing with the control group of which expression level of MHC and myoD was set to 1. (See Table 1).

[0134] As shown in FIG. 2, the effect of test sample on the muscle differentiation was determined according to immunofluorescence staining method using by anti-MHC antibody and DAPI (4′,6-diamidino-2-phenylindole). The cells added with various concentrations of inventive extract (1, 10, 100 ng/ml) were treated to myotubes differentiation medium to be differentiate to muscle fiber for 3 days. The muscle fibers were collected and the level of differentiation markers to muscle fibers, i.e., MHC and myoD protein was analyzed according to Western blot analysis.

[0135] Increased red-fluorescence means that the inventive extract promotes MHC expression in C2C12 cells in a concentration-dependent manner, and it has been confirmed that cylinder-shaped myoblast cells expressing MHC are found to be multinucleated through DAPI counter staining method (See Table 2).

[0136] In this experiment, it has been confirmed that inventive extract increases the expression of MHC and myoD protein in myotube cells and the number of cylinder-shaped multinucleated myotubes, resulting in promoting muscle cell differentiation (See Table 1-2, FIG. 1-2).

TABLE-US-00001 TABLE 1 Effect of inventive extract on the expression of MHC and myoD (See FIG. 1) AJE (ng/mL) concentration 0 1 10 100 MHC/pan-cadherin 1.0 1.3 1.4 1.6 expression (fold) myoD/pan-cadherin 1.0 1.8 1.7 1.5 expression (fold)

TABLE-US-00002 TABLE 2 Effect of inventive extract on the expression of MHC in cylinder-shaped multinucleated myotubes (See FIG. 2) AJE (ng/mL) concentration 0 1 10 100 The number of muscle fibers that are 1 3.3 5.2 5.2 multinucleated with five or more nuclei and expressed with MHC (fold)

Experimental Example 2. Study on p38 MAPK Signaling System Mechanism of Inventive Extract for Promoting Myoblast Differentiation

[0137] In order to determine the effect of the inventive extract obtained in Examples on p38 MAPK signaling system mechanism for promoting myoblast differentiation, following experiment was performed according to the procedure disclosed in the literature (Mol. Biol. Cell. 2010, 21:2399; Trends Cell Biol. 2006, 16:36).

[0138] Following test has been performed according to the procedure disclosed in the literature to confirm whether the inventive extract takes an effect to p38 MAPK signaling system mechanism during myoblast differentiation or not since it has been reported that p38 MAPK signaling system mechanism is very important to promote myoblast differentiation (See FIG. 2) (Mol. Biol. Cell. 2010, 21:2399).

[0139] p38 mitogen-activated protein kinase (p38 MAPK) plays an important role in MyoD activation, and p38MAP promotes the dimerization of MyoD to induce the expression of myogenic factors (Trends Cell Biol. 2006, 16:36).

[0140] 2-1. Test Procedure

[0141] Various concentrations of inventive extract (1, 10, 100 ng/ml) were treated to myoblast cells and the expressed level of p38 MAPK protein in the lysates of differentiated myotubes obtained at 3 days after the differentiation, was determined according to Western blot analysis.

[0142] Primary rabbit-anti phosphorylation p38 MAPK (Cat #9212, Cell Signaling Technology) was reacted with anti-rabbit secondary antibody (goat anti-rabbit IgG-HRP, sc-2004, SantaCruz) to determine the express level of phosphorylated p38 MAPK and primary rabbit-anti-p38 MAPK (Cat #9212, Cell Signaling Technology) was used to determine the expressed amount of total p38 MAPK (loading control)

[0143] 2-2. Test Result (Table 3 and FIG. 3)

[0144] It has been confirmed that p38 MAPK signaling system, which is important for myoblast cell differentiation, was further activated during differentiation by the treatment of inventive samples through Western assays which confirms the promoting effect of inventive extract on p38 MAPK (See Table 3).

[0145] The above test result confirm that the inventive extract promotes the myoblast differentiation through p38 MAPK activation mechanism.

TABLE-US-00003 TABLE 3 Effect of inventive extract on p38 MAPK activity (FIG. 3) AJE (ng/mL) Concentration 0 1 10 100 phosphorylated-p38/p38 1.0 5.9 9.0 5.7 expressed amount (fold)

Experimental Example 3. Stimulation Effect of Inventive Compounds on the Initiation Effect of Myoblast Differentiation

[0146] In order to investigate the myogenic effect of the inventive compounds 1-6 obtained in Examples on the myoblast differentiation, following experiment was performed according to the procedure disclosed in the previous literature (Chem. Biol. Interact. 2016, 248:60).

[0147] 3-1. Test Procedure

[0148] Various inventive compounds (10 nM) were treated to C2C12 cells (mouse myoblast cell line; Cat #CRL-1771, American Type Culture Collection (ATCC)) and cell lysates obtained from the differentiated cells, were subjected to Western Blot analysis to evaluate the expressed level of MHC proteins (The result was shown in FIG. 4). the change of MHC expression caused by inventive compounds in myotube cell was determined by immunostaining method using by mouse anti-MHC and anti-mouse IgG2b Alexa-fluor 568 (See FIG. 5) (Chem. Biol. Interact. 2016, 248:60).

[0149] Various inventive compounds (10 nM) were treated to C2C12 cells (mouse myoblast cell line; Cat.sup.#CRL-1771, ATCC) in differentiation medium (2% horse serum-containing DMEM, Cat.sup.#11965-084, Gibco) for 3 days to induce their differentiation.

[0150] 3-1-1. Western Blot Analysis

[0151] For Western blot analysis, about 3×10.sup.4 mouse myoblast cell cells were incubated in 60 mm plates for 24 hours, and each sample was added to the differentiation medium for 3 days to differentiate. The protein extract was obtained from the differentiated cells using by lysis buffer [25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail, Calbiochem, Darmstadt, Germany). The protein extract (25 μg) was performed to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes.

[0152] Primary mouse anti-MHC (sc-376157, Santa Cruz) or anti-myoD (sc-32758, Santa Cruz) was conjugated to the blots at 4° C., for 12 hrs and then conjugated with anti-mouse secondary antibody conjugated to Horseradish peroxidase (goat anti-mouse IgG-HRP, sc-2005, SantaCruz) to analyze the amount of MHC protein by chemiluminescence method. Pan-cadherin (Cat #3678, Sigma) was used as a loading control in the experiment.

[0153] 3-1-2. Immunofluorescence Staining

[0154] According to the above-described method, the differentiated myoblast cells in the differentiation medium were subject to immunofluorescence staining method.

[0155] After removing respective differentiation medium, the differentiated myoblast cells in the differentiation medium were washed with phosphate buffer solution twice and fixed with 4% paraformaldehyde (0141, BBC Biochemical) for 20 mins. After washing the cells with phosphate buffer solution twice again, the cells were treated with 0.1% tritonX-100 (2315025, Sigma) for 20 mins. After washing the cells with phosphate buffer solution twice again, the cells were blocked with 5% horse serum solution (16050122, Gibco) and mouse anti-MHC (MAB4470, R&D systems) was added thereto to react for 12 hours at 4° C.

[0156] After finishing the reaction, the cells were washed with phosphate buffer solution more than 3 times and then the level of MHC expression was analyzed using by Alexa Flouor 568-conjuagted secondary anti-mouse (A-21144, MicoProbes) and DAPI (D9542, Sigma). The test result of immunofluorescence on MHC-positive myotubes was visualized with red color and the DAPI-labelled nucleus was visualized with blue color.

[0157] 3-2. Test Result (See Table 4 and FIG. 4-5)

[0158] As shown in FIG. 4, the effect of test sample (inventive compounds 1-6) on the expression of MHC and myoD was determined. The cells added with various concentrations of inventive compounds (10 nM) were treated to myotubes differentiation medium to be differentiate to muscle fiber for 3 days. The muscle fibers were collected and the level of differentiation markers to muscle fibers, i.e., MHC protein was analyzed according to Western blot analysis.

[0159] At the result, it can be confirmed that the test sample group treated with inventive compounds showed increased expression level comparing with the control group of which expression level of MHC was set to 1. (See Table 4).

[0160] As shown in FIG. 5, the effect of test sample on the muscle differentiation was determined according to immunofluorescence staining method using by anti-MHC antibody and DAPI (4′,6-diamidino-2-phenylindole). The cells added with inventive compounds (10 nM) were treated to myotubes differentiation medium to be differentiate to muscle fiber for 3 days. The muscle fibers were collected and the level of differentiation markers to muscle fibers, i.e., MHC and myoD protein was analyzed according to Western blot analysis.

[0161] Increased red-fluorescence means that the inventive compounds promote MHC expression in C2C12 cells in a concentration-dependent manner, and it has been confirmed that cylinder-shaped myoblast cells expressing MHC are found to be multinucleated through DAPI counter staining method.

[0162] In this experiment, it has been confirmed that inventive compounds 1-6 increases the expression of MHC protein in myotube cells and the number of cylinder-shaped multinucleated myotubes, resulting in promoting muscle cell differentiation (See Table 4-5, FIG. 4-5).

TABLE-US-00004 TABLE 4 Effect of inventive compounds on the expression of MHC and myoD (See FIG. 4) compound (10 nM) — 1 2 3 4 5 6 MHC/pan-cadherin 1.0 1.5 1.7 1.6 1.9 1.9 1.5 expression (fold)

TABLE-US-00005 TABLE 5 Effect of inventive compounds on the expression of MHC in cylinder-shaped multinucleated myotubes (See FIG. 5) Compound (10 nM) — 1 2 3 4 5 6 The number of muscle fibers 1.0 2.8 3.2 3.1 3.1 3.2 2.9 that are multinucleated with five or more nuclei and expressed with MHC (fold)

Experimental Example 4. Promoting Effect of Inventive Compound on Myoblast Differentiation

[0163] In order to investigate the promoting effect of the inventive compound 1 obtained in Examples on myoblast differentiation, following experiment was performed according to the procedure disclosed in the previous literature (Chem. Biol. Interact. 2016, 248:60).

[0164] 4-1. Test Procedure

[0165] In order to investigate the myogenic effect of the inventive compound 1 obtained in Example, various concentrations of inventive compound 1 (1, 10 and 100 nM) were treated to C2C12 cells (mouse myoblast cell line; Cat.sup.#CRL-1771, American Type Culture Collection (ATCC)) and cell lysates obtained from the differentiated cells, were subjected to Western Blot analysis to evaluate the expressed level of MHC and myoD proteins (The result was shown in FIG. 6). The change of MHC expression caused by inventive compound in myotube cell was determined by immunostaining method using by mouse anti-MHC and anti-mouse IgG2b Alexa-fluor 568 (See FIG. 7) (Chem. Biol. Interact. 2016, 248:60).

[0166] Various concentrations of inventive compound 1 (1, 10 and 100 nM) were treated to C2C12 cells (mouse myoblast cell line; Cat.sup.#CRL-1771, ATCC) in differentiation medium (2% horse serum-containing DMEM, Cat.sup.#11965-084, Gibco) for 3 days to induce their differentiation.

[0167] 4-1-1. Western Blot Analysis

[0168] For Western blot analysis, about 3×10.sup.4 mouse myoblast cell cells were incubated in 60 mm plates for 24 hours, and each sample was added to the differentiation medium for 3 days to differentiate. The protein extract was obtained from the differentiated cells using by lysis buffer [25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail, Calbiochem, Darmstadt, Germany). The protein extract (25 μg) was performed to SDS-polyacrylamide gel electorphoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes.

[0169] Primary mouse anti-MHC (sc-376157, Santa Cruz) or anti-myoD (sc-32758, Santa Cruz) was conjugated to the blots at 4° C., for 12 hrs and then conjugated with anti-mouse secondary antibody conjugated to Horseradish peroxidase (goat anti-mouse IgG-HRP, sc-2005, SantaCruz) to analyze the amount of MHC protein by chemiluminescence method. Pan-cadherin (Cat #3678, Sigma) was used as a loading control in the experiment.

[0170] 4-1-2. Immunofluorescence Staining

[0171] According to the above-described method, the differentiated myoblast cells in the differentiation medium were subject to immunofluorescence staining method.

[0172] After removing respective differentiation medium, the differentiated myoblast cells in the differentiation medium were washed with phosphate buffer solution twice and fixed with 4% paraformaldehyde (0141, BBC Biochemical) for 20 mins. After washing the cells with phosphate buffer solution twice again, the cells were treated with 0.1% tritonX-100 (2315025, Sigma) for 20 mins. After washing the cells with phosphate buffer solution twice again, the cells were blocked with 5% horse serum solution (16050122, Gibco) and mouse anti-MHC (MAB4470, R&D systems) was added thereto to react for 12 hours at 4° C.

[0173] After finishing the reaction, the cells were washed with phosphate buffer solution more than 3 times and then the level of MHC expression was analyzed using by Alexa Flouor 568-conjuagted secondary anti-mouse (A-21144, MicoProbes) and DAPI (D9542, Sigma). The test result of immunofluorescence on MHC-positive myotubes was visualized with red color and the DAPI-labelled nucleus was visualized with blue color.

[0174] 4-2. Test Result (See Table 6 and FIG. 6-7)

[0175] As shown in FIG. 6, the effect of test sample (inventive compounds 1) on the expression of myoD was determined. The cells added with various concentrations of inventive compound 1 (1, 10, 100 nM) were treated to myotubes differentiation medium to be differentiate to muscle fiber for 3 days. The muscle fibers were collected and the level of differentiation markers to muscle fibers, i.e., MHC and myoD protein were analyzed according to Western blot analysis.

[0176] At the result, it can be confirmed that the test sample group treated with inventive compound 1 showed increased expression level comparing with the control group of which expression level of MHC and myoD was set to 1. (See Table 6).

[0177] As shown in FIG. 7, the effect of compound 1 on the muscle differentiation was determined according to immunofluorescence staining method using by anti-MHC antibody and DAPI (4′,6-diamidino-2-phenylindole).

[0178] Increased red-fluorescence means that the inventive compound 1 promotes MHC expression in C2C12 cells in a concentration-dependent manner, and it has been confirmed that cylinder-shaped myoblast cells expressing MHC are found to be multinucleated through DAPI counter staining method (See Table 7).

[0179] In this experiment, it has been confirmed that inventive compound 1 increases the expression of MHC and myoD proteins in myotube cells and the number of cylinder-shaped multinucleated myotubes, resulting in promoting muscle cell differentiation (See Table 6-7, FIG. 6-7).

TABLE-US-00006 TABLE 6 Effect of inventive compound 1 on the expression of MHC and myoD (See FIG. 6) compound 1(nM) concentration 0 1 10 100 MHC/pan-cadherin 1.0 1.2 6.0 4.6 expression (fold) myoD/pan-cadherin 1.0 1.2 1.3 1.7 expression (fold)

TABLE-US-00007 TABLE 7 Effect of inventive compound 1 on the expression of MHC in cylinder-shaped multinucleated myotubes (See FIG. 7) compound 1 (nM) concentration 0 1 10 100 The number of muscle fibers that are 1 1.5 9.0 8.0 multinucleated with five or more nuclei and expressed with MHC (fold)

Experimental Example 5. Study on p38 MAPK Signaling System Mechanism of Inventive Compound 1 for Promoting Myoblast Differentiation

[0180] In order to determine the effect of the inventive compound 1 obtained in Examples on p38 MAPK signaling system mechanism for promoting myoblast differentiation, following experiment was performed according to the procedure disclosed in the literature (Mol. Biol. Cell. 2010, 21:2399; Trends Cell Biol. 2006, 16:36).

[0181] Following test has been performed according to the procedure disclosed in the literature to confirm whether the inventive compound 1 takes an effect to p38 MAPK signaling system mechanism during myoblast differentiation or not since it has been reported that p38 MAPK signaling system mechanism is very important to promote myoblast differentiation (Mol. Biol. Cell. 2010, 21:2399).

[0182] 5-1. Test Procedure

[0183] Various concentrations of inventive compound 1 (1, 10, 100 nM) were treated to myoblast cells and the expressed level of p38 MAPK protein in the lysates of differentiated myotubes obtained at 3 days after the differentiation, was determined according to Western blot analysis.

[0184] Primary rabbit-anti phosphorylation p38 MAPK (Cat #9212, Cell Signaling Technology) was reacted with anti-rabbit secondary antibody (goat anti-rabbit IgG-HRP, sc-2004, SantaCruz) to determine the express level of phosphorylated p38 MAPK and primary rabbit-anti-p38 MAPK (Cat #9212, Cell Signaling Technology) was used to determine the expressed amount of total p38 MAPK (loading control)

[0185] 5-2. Test Result (Table 8 and FIG. 8)

[0186] It has been confirmed that p38 MAPK signaling system, which is important for myoblast cell differentiation, was further activated during differentiation by the treatment of inventive samples through Western assays which confirms the promoting effect of inventive compound 1 on p38 MAPK (See Table 8).

[0187] The above test result confirm that the inventive test sample promotes the myoblast differentiation through p38 MAPK activation mechanism.

TABLE-US-00008 TABLE 8 Effect of inventive compound 1 on p38 MAPK activity (FIG. 8) Compound 1 (nM) Concentration 0 1 10 100 phosphorylated-p38/p38 1.0 10.6 14.8 15.0 expressed amount (fold)

Experimental Example 6. Inhibitory Effect of Inventive Extract on Muscle Loss

[0188] In order to confirm the inhibitory effect on muscle loss of the inventive extract obtained in Examples, following experiment was performed according to the procedure disclosed in the previous literature (Int. J. Mol. Med. 2015, 36:29; Biomed. Pharmacother. 2017, 95:1486)

[0189] 6-1. Test Procedure

[0190] In order to confirm the inhibitory effect of the inventive extract obtained in Example, dexamethasone was treated to myotube cell to establish in vitro model of muscle loss and following experiment was performed according to the procedure disclosed in the previous literature (Int. J. Mol. Med. 2015, 36:29).

[0191] In particular, following experiment was performed according to the procedure disclosed in the previous literature to find out whether inventive extract takes an effect on MAFbx expression during muscle protein degradation by dexamethasone treatment since the activity of MAFbx, a muscle proteolytic enzyme, is known to induce muscle protein loss (Biomed. Pharmacother. 2017, 95:1486)

[0192] 6-1-1. Western Blot Analysis

[0193] For Western blot analysis, about 3×10.sup.4 mouse myoblast cell cells (C2C12; CRL-1772 ATCC) were incubated in 60 mm plates for 24 hours, and various concentrations of each inventive extract (0, 1, 10, 100 ng/mL) was added to the differentiation medium for 3 days to differentiate. The protein extract was obtained from the differentiated cells using by lysis buffer [25 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), and protease inhibitor cocktail, Calbiochem, Darmstadt, Germany). The protein extract (25 μg) was performed to SDS-polyacrylamide gel electorphoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes.

[0194] Primary mouse anti-MHC (sc-376157, Santa Cruz) or primary mouse anti-MAFbx (sc-166806, Santa Cruz) was conjugated to the blots at 4° C., for 12 hrs and then conjugated with anti-mouse secondary antibody conjugated to Horseradish peroxidase (goat anti-mouse IgG-HRP, sc-2005, Santa Cruz) to analyze the amount of MHC and MAFbx protein by chemiluminescence method. Pan-cadherin (Cat #3678, Sigma) was used as a loading control in the experiment.

[0195] 6-1-2. Immunofluorescence Staining

[0196] According to the above-described method, the differentiated myoblast cells in the differentiation medium were subject to immunofluorescence staining method.

[0197] After removing respective differentiation medium, the differentiated myoblast cells in the differentiation medium were washed with phosphate buffer solution twice and fixed with 4% paraformaldehyde (0141, BBC Biochemical) for 20 mins. After washing the cells with phosphate buffer solution twice again, the cells were treated with 0.1% tritonX-100 (2315025, Sigma) for 20 mins. After washing the cells with phosphate buffer solution twice again, the cells were blocked with 5% horse serum solution (16050122, Gibco) and mouse anti-MHC (MAB4470, R&D systems) was added thereto to react for 12 hours at 4° C.

[0198] After finishing the reaction, the cells were washed with phosphate buffer solution more than 3 times and then the level of MHC expression was analyzed using by Alexa Flouor 568-conjuagted secondary anti-mouse (A-21144, MicoProbes) and DAPI (D9542, Sigma). The test result of immunofluorescence on MHC-positive myotubes was visualized with red color and the DAPI-labelled nucleus was visualized with blue color.

[0199] 6-2. Test Result

[0200] As a result of analyzing MHC protein expression in each cell group through Western blotting assays, when MHC expression in negative control group (NC), i.e., myotube cells that had not treated with dexamethasone, was set to 1, that in the positive control group, i.e., myotube cells treated with dexamethasone, showed the reduced level to 0.7 fold and that in test sample group i.e., myotube cells treated with inventive test sample, also showed recovered level.

[0201] Additionally, it has been confirmed that when MAFbx expression in negative control group (NC), i.e., myotube cells that had not treated with dexamethasone, was set to 1, that in the positive control group, i.e., myotube cells treated with dexamethasone to indue muscle loss, showed the increased level to 1.9 fold, and the level had been decreased to 0.3 fold in a dose dependent manner in test sample group i.e., myotube cells treated with inventive test sample, which means the inhibitory effect of inventive extract on proteolysis of myoprotein (See Table 9 and FIG. 9-10).

[0202] As shown in FIG. 9, MHC expression was evaluated at the level of red fluorescence in myotube cells to which both of inventive test sample and dexamethasone were added, and the formation of multinucleated myotubes induced by treatment with inventive test samples was determined using by DAPI reading staining method.

[0203] It has been confirmed that the group treated with dexamethasone showed increased loss of myotube cell whereas the test group treated with test sample strong inhibitory effect on the loss of multinucleated MHC-positive cells caused by dexamethasone treatment (Tables 10 and FIG. 10).

[0204] Accordingly, it has been demonstrated that the inventive test sample strong protective activity from muscle loss.

TABLE-US-00009 TABLE 9 protecting activity of inventive extract on myotube cells (FIG. 9) Control AJE (ng/mL) group 0 1 10 100 MHC/pan-cadherin 1.0 0.9 1.3 1.4 1.6 expression (fold) MAFbx/pan-cadherin 1.0 1.9 0.3 0.3 0.6 expression (fold)

TABLE-US-00010 TABLE 10 Effect of inventive extract on the expression of MHC in cylinder-shaped multinucleated myotubes (See FIG. 10) Control AJE (ng/mL) group 0 1 10 100 The number of muscle fibers that are 1.0 0.1 0.7 0.9 0.8 multinucleated with five or more nuclei and expressed with MHC (fold)

Experimental Example 7. Inhibitory Effect of Inventive Compounds on Muscle Loss

[0205] In order to confirm the inhibitory effect on muscle loss of the inventive compounds obtained in Examples, following experiment was performed according to the procedure disclosed in the previous literature (Int. J. Mol. Med. 2015, 36:29; Biomed. Pharmacother. 2017, 95:1486)

[0206] 7-1. Test Procedure

[0207] In order to confirm the inhibitory effect of the inventive compounds obtained in Example, dexamethasone was treated to myotube cell to establish in vitro model of muscle loss and following experiment was performed according to the procedure disclosed in the previous literature (Int. J. Mol. Med. 2015, 36:29).

[0208] About 3×10.sup.4 mouse myoblast cell cells (C2C12; CRL-1772 ATCC) were incubated in 60 mm plates for 24 hours, and various inventive compounds (10 nM) were added to the differentiation medium (0273, Gibco) for 3 days to differentiate.

[0209] In order to establish in vitro model of muscle loss, 1 mM dexamethasone (D4902, Sigma) was treated to each differentiated myotube cell for 12 hours to differentiate and the cell was washed with phosphate buffer solution to perform immunofluorescence staining in order to confirm MHC expression (See FIG. 11).

[0210] 7-1-1. Immunofluorescence Staining

[0211] According to the above-described method, the differentiated myoblast cells in the differentiation medium were subject to immunofluorescence staining method.

[0212] After removing respective differentiation medium, the differentiated myoblast cells in the differentiation medium were washed with phosphate buffer solution twice and fixed with 4% paraformaldehyde (0141, BBC Biochemical) for 20 mins. After washing the cells with phosphate buffer solution twice again, the cells were treated with 0.1% tritonX-100 (2315025, Sigma) for 20 mins. After washing the cells with phosphate buffer solution twice again, the cells were blocked with 5% horse serum solution (16050122, Gibco) and mouse anti-MHC (MAB4470, R&D systems) was added thereto to react for 12 hours at 4° C.

[0213] After finishing the reaction, the cells were washed with phosphate buffer solution more than 3 times and then the level of MHC expression was analyzed using by Alexa Flouor 568-conjuagted secondary anti-mouse (A-21144, MicoProbes) and DAPI (D9542, Sigma). The test result of immunofluorescence on MHC-positive myotubes was visualized with red color and the DAPI-labelled nucleus was visualized with blue color.

[0214] 7-2. Test Result

[0215] The MHC expression was evaluated at the level of red fluorescence in myotube cells to which both of inventive test sample and dexamethasone were added, and the formation of multinucleated myotubes induced by treatment with inventive test samples was determined using by DAPI reading staining method.

[0216] It has been confirmed that the group treated with dexamethasone showed increased loss of myotube cell whereas the test group treated with test sample strong inhibitory effect on the loss of multinucleated MHC-positive cells caused by dexamethasone treatment (Tables 11 and FIG. 11).

[0217] Accordingly, it has been demonstrated that the inventive test sample strong protective activity from muscle loss.

TABLE-US-00011 TABLE 11 Effect of inventive compounds on the expression of MHC in cylinder-shaped multinucleated myotubes in dexamethasone- induced loss of myotube cell (See FIG. 11) Control compound (10 nM) group 0 1 2 3 4 5 6 The number of muscle fibers 1.0 0.4 0.7 0.8 0.5 0.7 0.8 0.7 that are multinucleated with five or more nuclei and expressed with MHC (fold)

Experimental Example 8. Improving Effect of Inventive Extract on Motility in Animal Model of Muscle Loss (In Vivo)

[0218] In order to confirm the improving effect on motility in animal model of muscle loss of the inventive compounds obtained in Examples, following experiment was performed according to the procedure disclosed in the previous literature (Int. J. Mol. Med. 2015, 36:29; Biomed. Pharmacother. 2017, 95:1486)

[0219] Dexamethasone, a synthetic glucocorticoid, was injected subcutaneously into C57/BL6 mouse mice to establish an animal model of muscle loss and following experiment was performed.

[0220] 8-1. Test Procedure

[0221] In order to confirm the inhibitory effect of the inventive compounds obtained in Example, dexamethasone

[0222] To establish an in vivo model of muscle loss, 6 weeks-old male C57/BL6 mice (Raon Bio, 6 weeks old, weight range: 32-36 g) were acclimated for one-week, and then weighed animals determined to be healthy during the acclimatization period. The mice close to the average weight were selected using a simple random sampling method and were classified into five groups, namely, (a) a control group, (b) a muscle loss control group (treated with dexamethasone 1 mg/kg), (c) test sample group treated with inventive extract 250 mg/kg and dexamethasone, (d) test sample group treated with inventive extract 500 mg/kg and dexamethasone, and (e) positive control treated with oxymetholone (Celltrion Co. Ltd, ATC #A14AA05, 10 mg/kg) and dexamethasone.

[0223] The inventive Extract and oxymetholone were administered once to the mice daily for 4 weeks and dexamethasone was injected subcutaneously for 10 days from 10 days before the end of co-treatment of inventive extract and oxymetholone.

[0224] Both of inventive extract and oxymetholone were forcibly administered orally using zonde for oral administration after holding and fixing the skin of the animal's poll area, and dexamethasone was injected slowly subcutaneously after inserting a syringe into the dorsal skin fold of the animal held on the thumb and index finger, slightly aspirating whether it was injected normally, and confirming that no blood came out.

[0225] 8-1-1. Determination of Body Weight

[0226] The body weight of every animal was measured once a week for 4 weeks from the day of inventive extract administration.

[0227] 8-1-2. Grip Strength Test

[0228] The grip strength of mice was determined as follows; after grabbing the animal's tail and placing it on the grid, the front foot and body were pulled at the same speed until the front foot and body were held at the grid with the front paw, and the average value was calculated after three experimental repetitions. The determination was performed on the 14th and 28th days after the administration of test sample.

[0229] 8-1-3. Treadmill Test

[0230] The treadmill test of mice was determined as follows; the speed of treadmill was set to 12 m/min as a starting speed, and the speed was gradually increased by 3 m per minute to reach to 30 m/min as a final speed, and the test was measured for a total of 30 minutes. The determination was performed on the 14th and 28th days after the administration of test sample.

[0231] 8-1-4. Measurement of Muscle Mass, Epididymal Fat, and Liver Weight in Animal

[0232] The treadmill test of mice was determined as follows; the speed of treadmill was set to 12 m/min as a starting speed, and the speed was gradually increased by 3 m per minute to reach to 30 m/min. The mice were anesthetized with anesthetic to prevent the movement of experimental animals and sacrificed to minimize pain to dissect them. All instruments used during the experiment were sterilized and disinfected and their liver and epididymis fat were harvested.

[0233] The gastrocnemius muscle and soleus muscle of the mice were separated, washed in saline, drained, and weighed.

[0234] 8-2. Test Result (Tables 12-16)

[0235] In order to confirm the inhibitory effect of the inventive test samples obtained in Example, dexamethasone

[0236] 8-2-1. Change in Body Weight

[0237] Compared to the control group, there was no change in body weight in the sole treatment group with dexamethasone alone, and the group treated with inventive extract with 250 mg/kg and 500 mg/kg showed significant weight loss compared to the control group (See Table 12).

[0238] 8-2-2. Motility Improving Activity

[0239] At the test result of grip strength measurements at week 4, it has been confirmed that the control group treated with dexamethasone showed decreased grip strength whereas the test group treated with 250 mg/kg and 500 mg/kg of inventive extract and positive control group showed significantly increased grip strength, which indicates that the inventive extract has the potential to improve muscle strength loss caused by muscular dystrophy. (See Table 12)

[0240] At the test result of 4 treadmill assessment at week 4, it has been confirmed that the group treated with dexamethasone showed decreased motility compared to the control group, and both of the test group treated with inventive extract and positive control group showed a tendency to increase motility reduced by dexamethasone treatment, of which difference was not significant. (See Table 14).

[0241] In summary, it has been confirmed that the inventive extract has the possibility to improve endurance-related motility activity.

[0242] 8-2-3. Absolute Change in the Weight of Muscle Mass, Epididymal Fat, and Liver Weight in Animal (Table 15)

[0243] After autopsy at 4 weeks after administration, it has been confirmed that the groups showed no significant difference between the groups in respect of the weight of liver and epididymis fat while the test group treated with inventive extract (500 mg/kg) and positive control group showed significant decrease compared to dexamethasone treatment group.

[0244] As a result of the weighing of calf muscle, there was a significant decrease in the dexamethasone treatment group compared to the control group, while there was no significant change in either the test sample group treated with inventive extract or the positive control group compared to the dexamethasone treatment group.

[0245] As a result of the weighing of fluke muscle, the group treated with dexamethasone showed significant decrease compared to the control group, while both of test sample group treated with inventive extract 250 mg/kg or 500 mg/kg and positive control group showed significant increase compared to the dexamethasone treatment group.

[0246] As a result, it was confirmed that muscle loss by dexamethasone significantly can be increased by the treatment of inventive extract in a dose dependent manner.

[0247] 8-2-4. Relative Change in the Weight of Muscle Mass, Epididymal Fat, and Liver Weight in Animal (Table 16)

[0248] After autopsy at 4 weeks after administration, the weight of liver and Epididymis fat was weighed to calculate weight-to-weight to compare the relative differences between the organ's weight in the groups.

[0249] It has been confirmed that the groups showed no significant difference between the groups in respect of the weight of liver and epididymis fat.

[0250] As a result of the weighing of calf muscle, there was a significant decrease in the dexamethasone treatment group compared to the control group, while there was significant increase in the test sample group treated with inventive extract (500 mg/kg) compared to the dexamethasone treatment group.

[0251] As a result of the weighing of fluke muscle, the group treated with dexamethasone showed significant decrease compared to the control group, while both of test sample group treated with inventive extract 250 mg/kg or 500 mg/kg and positive control group showed significant increase compared to the dexamethasone treatment group.

[0252] As a result, it was confirmed that muscle loss by dexamethasone significantly can be increased by the treatment of inventive extract in a dose dependent manner.

[0253] In summary, it has been confirmed that the inventive extract has the improving activity of motor capacity decline and muscle loss due to muscular atrophy under this test condition.

TABLE-US-00012 TABLE 12 Effect of inventive extract on the change in body weight (unit: gram) groups 0 week 1 week 2 weeks 3 weeks 4 weeks Control 22.47 ± 0.82 23.17 ± 1.10 23.78 ± 1.18 24.08 ± 1.16 24.46 ± 0.98.sup.a Dexa 22.33 ± 1.11 22.84 ± 1.54 23.46 ± 1.60 23.57 ± 1.81  23.57 ± 1.76.sup.ab Dexa + 250 22.31 ± 1.04 22.28 ± 0.82 22.70 ± 0.71 22.94 ± 0.93 22.74 ± 0.92.sup.b Dexa + 500 22.33 ± 1.10 22.51 ± 1.44 23.23 ± 1.07 22.60 ± 1.04 22.57 ± 1.29.sup.b Dexa + Oxy 22.26 ± 0.71 22.49 ± 0.72 23.25 ± 0.90 23.22 ± 0.93 23.20 ± 0.78.sup.b .sup.a, bsignificance between the different characters (p < 0.01) Dexa: dexamethasone treatment group Oxy: oxymetholone

TABLE-US-00013 TABLE 13 Effect of inventive extract on the change in grip strength (unit: N) groups 2 weeks 4 weeks Control 1.09 ± 0.15 1.23 ± 0.12.sup.a Dexa 1.05 ± 0.08 0.94 ± 0.12.sup.b Dexa + 250 1.01 ± 0.10 1.08 ± 0.11.sup.c Dexa + 500 1.04 ± 0.12 1.26 ± 0.07.sup.a Dexa + Oxy 1.00 ± 0.06 1.25 ± 0.16.sup.a .sup.a, b, csignificance between the different characters (p < 0.01) Dexa: dexamethasone treatment group Oxy: oxymetholone

TABLE-US-00014 TABLE 14 Effect of inventive extract on the motility (unit: meter) groups 2 weeks 4 weeks Control 776.98 ± 49.08 784.83 ± 38.13 Dexa 798.85 ± 14.33  745.73 ± 30.01* Dexa + 250 793.93 ± 34.29 763.40 ± 37.72 Dexa + 500 802.38 ± 27.62 783.55 ± 26.28 Dexa + Oxy 774.75 ± 66.11 773.93 ± 39.64 *significance compared to control group (p < 0.01) Dexa: dexamethasone treatment group Oxy: oxymetholone

TABLE-US-00015 TABLE 15 absolute change in the weight of muscle mass, epididymal fat, and liver weight in animal (unit: gram) groups liver epididymal fat calf muscle fluke muscle Control 1.16 ± 0.11 0.50 ± 0.09 0.270 ± 0.019   0.019 ± 0.002.sup.ab Dexa 1.15 ± 0.13 0.51 ± 0.10 0.242 ± 0.014* 0.014 ± 0.002.sup.c Dexa + 250 1.11 ± 0.08 0.49 ± 0.10 0.246 ± 0.010* 0.017 ± 0.003.sup.b Dexa + 500 1.10 ± 0.09 .sup. 0.42 ± 0.05.sup.# 0.248 ± 0.007* 0.020 ± 0.002.sup.a Dexa + Oxy 1.16 ± 0.07 .sup. 0.43 ± 0.09.sup.# 0.251 ± 0.016* 0.023 ± 0.002.sup.d *significance compared to control group (p < 0.01), .sup.#significance compared to dexamethasone treatment group (p < 0.01) .sup.a, b, c, dsignificance between the different characters (p < 0.01) Dexa: dexamethasone treatment group Oxy: oxymetholone

TABLE-US-00016 TABLE 16 relative change in the weight of muscle mass, epididymal fat, and liver weight in animal (unit: gram) groups liver epididymal fat calf muscle fluke muscle Control 4.74 ± 0.33 2.04 ± 0.35 1.105 ± 0.070 0.077 ± 0.008.sup.a Dexa 4.88 ± 0.33 2.17 ± 0.30  1.031 ± 0.072* 0.060 ± 0.010.sup.b Dexa + 250 4.89 ± 0.28 2.17 ± 0.42 1.083 ± 0.043 0.075 ± 0.011.sup.a Dexa + 500 4.89 ± 0.34 1.86 ± 0.17 .sup. 1.104 ± 0.068.sup.# 0.091 ± 0.012.sup.c Dexa + Oxy 4.99 ± 0.29 1.86 ± 0.40 1.082 ± 0.061 0.099 ± 0.011.sup.c *significance compared to control group (p < 0.01), .sup.#significance compared to dexamethasone treatment group (p < 0.01) .sup.a, b, csignificance between the different characters (p < 0.01) Dexa: dexamethasone treatment group Oxy: oxymetholone

MODE FOR INVENTION

[0254] Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

Preparation of Powder

[0255] Compound 5: 20 mg [0256] Lactose: 100 mg [0257] Talc: 10 mg

[0258] Powder preparation was prepared by mixing above components and filling sealed package.

Preparation of Tablet

[0259] Compound 5: 10 mg [0260] Corn Starch: 100 mg [0261] Lactose: 100 mg [0262] Magnesium stearate: 2 mg

[0263] Tablet preparation was prepared by mixing above components and entabletting.

Preparation of Capsule

[0264] Compound 4: 10 mg [0265] Crystalline cellulose: 3 mg [0266] Lactose: 14.8 mg [0267] Magnesium stearate: 0.2 mg

[0268] Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

Preparation of Injection

[0269] Compound 3: 10 mg [0270] mannitol: 180 mg [0271] Distilled water for injection: 2974 mg [0272] Na.sub.2HPO.sub.4, 12H2O: 26 mg

[0273] Injection preparation was prepared by dissolving active component, and then filling all the components in 2 ml ample and sterilizing by conventional injection preparation method.

Preparation of Liquid

[0274] AJE extract: 20 mg [0275] Isomerization sugar: 10 g [0276] mannitol: 5 g [0277] distilled water: optimum amount

[0278] Liquid preparation was prepared by dissolving active component, and then filling all the components in 100m ample and sterilizing by conventional liquid preparation method.

Preparation of Health Food

[0279] AJE extract: 1000 mg [0280] Vitamin mixture: optimum amount [0281] Vitamin A acetate: 70 g [0282] Vitamin E: 1.0 mg [0283] Vitamin Bio: 13 mg [0284] Vitamin B.sub.2: 0.15 mg [0285] Vitamin B6: 0.5 mg [0286] Vitamin B1: 20.2 g [0287] Vitamin C: 10 mg [0288] Biotin: 10 g [0289] Amide nicotinic acid: 1.7 mg [0290] Folic acid: 50 g [0291] Calcium pantothenic acid: 0.5 mg [0292] Mineral mixture: optimum amount [0293] Ferrous sulfate: 1.75 mg [0294] Zinc oxide: 0.82 mg [0295] Magnesium carbonate: 25.3 mg [0296] Monopotassium phosphate: 15 mg [0297] Dicalcium phosphate: 55 mg [0298] Potassium citrate: 90 mg [0299] Calcium carbonate: 100 mg [0300] Magnesium chloride: 24.8 mg

[0301] The above mentioned vitamin and mineral mixture may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

Preparation of Health Beverage

[0302] Compound 2: 1000 mg [0303] Citric acid: 1000 mg [0304] Oligosaccharide: 100 g [0305] Apricot concentration: 2 g [0306] Taurine: 1 g [0307] Distilled water: 900 ml

[0308] Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85° C. for 1 hour, filtered and then filling all the components in 1000m ample and sterilizing by conventional health beverage preparation method.

[0309] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

INDUSTRIAL APPLICABILITY

[0310] As mentioned the above, the inventors of the present invention confirmed the treating effect of an extract of alder tree or the compounds isolated therefrom mentioned above on skeleton muscle disease through various in vitro test and animal model tests, for example, stimulation effect of inventive extract on the initiation effect of myoblast differentiation (Experimental Example 1, 3 and 4), confirming that inventive extract/compounds increases the number of cylinder-shaped multinucleated myotubes, resulting in promoting muscle cell differentiation; Study on p38 MAPK signaling system mechanism of inventive extract/compounds for promoting myoblast differentiation (Experimental Example 2 and 5); Inhibitory effect of inventive extract/compounds on muscle loss (Experimental Example 6 and 7); improving effect on motility in animal model of muscle loss (In vivo) (Experimental Example 8) confirming that the inventive extract/compounds promotes muscle cell differentiation as well as improves motor capacity decline and muscle loss. Therefore, the inventive extract/compounds of the present invention can be usefully used in a pharmaceutical composition, health functional food, and health supplement food for preventing and treating on skeleton muscle disease.