Methods for treating muscle wasting and degeneration diseases

Abstract

The present invention relates to a composition for preventing and treating muscle diseases or improving muscular function, containing, as an active ingredient, at least one selected from the group consisting of morusin, kuwanon G, and a Mori Cortex Radicis extract. The Mori Cortex Radicis extract, morusin, or kuwanon G, according to the present invention, has an effect of remarkably enhancing muscular function by increasing the expression of p-mTOR protein involved in muscular protein synthesis, inhibiting the expression of mRNAs of MuRF-1 and atrogin-1 involved in muscular protein degradation, and increasing the expression of mRNAs of MyoD and myogenin involved in muscular differentiation. In addition, the present invention is a natural product so as to be used safely without side effects, thereby being usable in drugs, food, or cosmetics.

Claims

1. A method for treating a muscle disease induced by muscle wasting or degeneration in a subject, the method comprising administering to the subject a composition consisting essentially of at least one compound selected from the group consisting of: (1) a compound of Formula 1: ##STR00012## (2) a compound of Formula 2 ##STR00013## and (3) an extract of Mori Cortex Radicis, wherein the compound is an active ingredient administered in an amount effective for enhancing muscle mass growth to treat the muscle disease induced by muscle wasting or degeneration, and wherein the muscle disease induced by muscle wasting or degeneration is selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscular degeneration, muscular spasticity, amyotrophic lateral sclerosis, myasthenia gravis, cachexia and sarcopenia.

2. The method of claim 1, wherein the composition is a pharmaceutical composition.

3. The method of claim 1, wherein the composition is a food composition or a cosmetic composition.

4. The method of claim 1, wherein the compound defined by Formula 1 or 2 is isolated from the extract of Mori Cortex Radicis.

5. The method of claim 1, wherein the Mori Cortex Radicis is a dried root bark of a plant belonging to a plant of family Moraceae and genus Morus spp.

6. The method of claim 1, wherein the extract of Mori Cortex Radicis is an extract obtained using at least one solvent selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid, a supercritical fluid, and a mixture thereof.

7. The method of claim 6, wherein the organic solvent having 1 to 6 carbon atoms is selected from the group consisting of alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, and petroleum ether.

8. The method of claim 1, wherein the extract of Mori Cortex Radicis is obtained by extracting Mori Cortex Radicis under an ultra-high pressure of 100 MPa to 1000 Mpa.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the results of measuring the expression of p-mTOR protein in L6 muscle cells after treating with morusin, kuwanon G, or the extract of Mori Cortex Radicis.

(2) FIG. 2 shows the results of measuring the expression of p-4EBP1 and p-p70S6K proteins after treating with morusin in L6 muscle cells.

(3) FIG. 3 shows the results of measuring the expression of p-4EBP1 and p-p70S6K proteins after treating with kuwanon G in L6 muscle cells.

(4) FIG. 4 shows the results of measuring the expression of p-p70S6K protein after treating with the hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Mori Cortex Radicis in L6 muscle cells.

(5) FIG. 5 shows the results of measuring the mRNA expression of MyoD and myogenin after treating with morusin in L6 muscle cells.

(6) FIG. 6 shows the results of measuring the mRNA expression of MyoD and myogenin after treating with kuwanon G in L6 muscle cells.

(7) FIG. 7 shows the results of measuring the mRNA expression of MyoD and myogenin by treating with the hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Mori Cortex Radicis in L6 muscle cells.

(8) FIG. 8 shows the results of measuring the mRNA expression of atrogin-1 and MuRF-1 by treating with morusin in L6 muscle cells.

(9) FIG. 9 shows the results of measuring the mRNA expression of atrogin-1 and MuRF-1 by treating with kuwanon G in L6 muscle cells.

(10) FIG. 10 shows the results of measuring the mRNA expression of atrogin-1 and MuRF-1 by treating with the hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Mori Cortex Radicis in L6 muscle cells.

MODE FOR CARRYING OUT INVENTION

(11) Hereinafter, the present invention will be described in more detail with reference to Examples.

(12) However, the following Examples are illustrative of the present invention, and the scope of the present invention is not limited to the following Examples.

Reference Example 1: Material Information of Morusin

(13) ##STR00008##

(14) Chemical Name: Morusin; 2-(2,4-Dihydroxyphenyl)-5-hydroxy-8,8-dimethyl-3-(3-methyl-2-buten-1-yl)-4H,8H-benzo[1,2-b:3,4-b]dipyran-4-one

(15) CAS No.: 62596-29-6

Reference Example 2: Material Information of Kuwanon G

(16) ##STR00009##

(17) Chemical Name: kuwanon G; 8-[(1S,5R,6S)-6-(2,4-Dihydroxybenzoyl)-5-(2,4-dihydroxyphenyl)-3-methyl-2-cyclohexen-1-yl]-2-(2,4-dihydroxyphenyl)-5,7-dihydroxy-3-(3-methyl-2-buten-1-yl)-4H-chromen-4-one

(18) CAS No.: 75629-19-5

Example 1

(19) Preparation of the Extract of Mori Cortex Radicis

(20) <1-1> Preparation of Methanol Extract of Mori Cortex Radicis

(21) The dried Mori Cortex Radicis was pulverized with a mixer, and 100 g of the pulverized Mori Cortex Radicis sample was added to 1 L of 100% methanol and extracted three times at room temperature for 24 hours. The extracted sample was filtered with Whatman No. 2 filter paper under reduced pressure, and the filtered extract was concentrated using a vacuum rotary condenser to remove the solvent component, and then a methanol extract of Mori Cortex Radicis was obtained.

(22) <1-2> Preparation of Ethanol Extract of Mori Cortex Radicis

(23) The dried Mori Cortex Radicis was pulverized with a mixer, and 100 g of the pulverized Mori Cortex Radicis sample was added to 1 L of 100% ethanol and extracted three times at room temperature for 24 hours. The extracted sample was filtered with Whatman No. 2 filter paper under reduced pressure, and the filtered extract was concentrated using a vacuum rotary condenser to remove the solvent component, and then an ethanol extract of Mori Cortex Radicis was obtained.

(24) <1-3> Preparation of Ethyl Acetate Extract of Mori Cortex Radicis

(25) The dried Mori Cortex Radicis was pulverized with a mixer, and 100 g of the pulverized Mori Cortex Radicis sample was added to 1 L of 100% ethyl acetate and extracted three times at room temperature for 24 hours. The extracted sample was filtered with Whatman No. 2 filter paper under reduced pressure, and the filtered extract was concentrated using a vacuum rotary condenser to remove the solvent component, and then an ethyl acetate extract of Mori Cortex Radicis was obtained.

(26) <1-4> Preparation of Hexane Extract of Mori Cortex Radicis

(27) The dried Mori Cortex Radicis was pulverized with a mixer, and 100 g of the pulverized Mori Cortex Radicis sample was added to 1 L of 100% hexane and extracted three times at room temperature for 24 hours. The extracted sample was filtered with Whatman No. 2 filter paper under reduced pressure, and the filtered extract was concentrated using a vacuum rotary condenser to remove the solvent component, and then a hexane of Mori Cortex Radicis was obtained.

(28) <1-5> Preparation of Hot Water Extract of Mori Cortex Radicis

(29) The root of the dried Mori Cortex Radicis was pulverized with a mixer, and 100 g of the pulverized Mori Cortex Radicis sample was added to 1 L of water and extracted while stirring at 80 C. for 2 hours. The extracted sample was filtered with Whatman No. 2 filter paper under reduced pressure, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components, and then a hot water extract of Mori Cortex Radicis was obtained.

(30) <1-6> Preparation of Ultra-High Pressure Extract of Mori Cortex Radicis

(31) The root of the dried Mori Cortex Radicis was pulverized with a mixer, 1 g of the pulverized Mori Cortex Radicis sample and 76 ml of 18% ethanol were placed in a polyethylene pack and sealed, and then extracted using an ultra-high pressure extraction apparatus (Frescal MFP-7000; Mitsubishi Heavy Industries, Tokyo, Japan). Extraction pressure was 320 MPa and extraction time was 5 min. The extracted sample was filtered with Whatman No. 2 filter paper, and the filtered extract was concentrated with a vacuum rotary condenser to remove solvent components to obtain an ultra-high pressure extract of Mori Cortex Radicis.

(32) <1-7> Preparation of Supercritical Fluid Extract of Mori Cortex Radicis

(33) The dried Mori Cortex Radicis was pulverized with a mixer, and 1 g of the pulverized Mori Cortex Radicis sample was filled into a sample cartridge and extracted with a supercritical fluid extraction apparatus (SFX 3560, Isco Inc., Lincoln, Nebr., USA). Supercritical fluid extraction conditions were extraction pressure 40 MPa, extraction temperature 50 C., supercritical carbon dioxide flow rate 60 mL/min, and extraction time 60 min. When the supercritical fluid extraction was completed, the supercritical fluid condition was released by lowering the pressure of the extraction device to obtain a supercritical fluid extract of Mori Cortex Radicis.

(34) <1-8> Preparation of Subcritical Fluid Extract of Mori Cortex Radicis

(35) The dried Mori Cortex Radicis was pulverized with a mixer, and 1 g of the pulverized Mori Cortex Radicis sample was added to 10 mL of distilled water and extracted with a subcritical fluid extractor (DIONEX Accelerated Solvent Extractor 100, DIONEX co., USA). The subcritical fluid extraction condition was extraction pressure 2.5 MPa, the extraction temperature 150 C. and the extraction time 15 minutes. The extracted sample was filtered with Wattman No. 2 filter paper, and the filtered extract was lyophilized at 40 C. to obtain a subcritical fluid extract of Mori Cortex Radicis.

Example 2

(36) Isolation of Morusin and Kuwanon G

(37) <2-1> Isolation of Morusin

(38) The dried Mori Cortex Radicis was pulverized with a mixer, and 100 g of the pulverized Mori Cortex Radicis sample was added to 1 L of 100% ethanol and extracted three times at room temperature for 24 hours. The extracted sample was filtered with Whatman No. 2 filter paper under reduced pressure, and the filtered extract was concentrated using a vacuum rotary condenser to remove the solvent component, and then an ethanol extract of Mori Cortex Radicis was obtained. The ethanol extract was loaded on a column packed with silica gel (silica gel; 70-230 mesh, Merck & Co., Whitehouse Station, N.J., USA), and then collected by using a solvent system mixed with hexane, ethyl acetate and methanol. The collected solution was then divided into eight lower fractions according to the above sorting order. The second fraction was concentrated again with a vacuum rotary evaporator to remove the solvent component. The concentrate was loaded on a column packed with RP-18, and then collected using a solvent system in which water, methanol, and acetonitrile were mixed. And the new collected solution was divided into five lower fractions according to the above sorting order. Morusin, a compound of the following Formula 1, was isolated and purified from fifth fractions:

(39) ##STR00010##

(40) <2-2> Isolation of Kuwanon G

(41) The dried Mori Cortex Radicis was pulverized with a mixer, and 100 g of the pulverized Mori Cortex Radicis sample was added to 1 L of 100% ethanol and extracted three times at room temperature for 24 hours. The extracted sample was filtered with Whatman No. 2 filter paper under reduced pressure, and the filtered extract was concentrated using a vacuum rotary condenser to remove the solvent component, and then an ethanol extract of Mori Cortex Radicis was obtained. The ethanol extract was loaded on a column packed with silica gel (silica gel; 70-230 mesh, Merck & Co., Whitehouse Station, N.J., USA), and then collected by using a solvent system mixed with hexane, ethyl acetate and methanol. And the collected solution was divided into eight lower fractions according to the above sorting order. The sixth fraction was again concentrated on a vacuum rotary evaporator to remove the solvent component. The concentrate was loaded on a column packed with RP-18, and then collected using a solvent system in which water, methanol, and acetonitrile were mixed. The new collected solution was then divided into six lower fractions according to the above sorting order. Kuwanon G, a compounds of Formula 2 were separated and purified in the third fractions:

(42) ##STR00011##

Example 3

(43) Effect of Morusin, Kuwanon G, and Mori Cortex Radicis on Muscle Formation

(44) L6 myoblasts (ATCC, Manassas, Va., USA) were added to a 6-well plate with Dulbecco's modified Eagle's Media (DMEM; Hyclone) containing 10% fetal bovine serum (FBS; Hyclone, Logan, Utah, USA) at a concentration of 210.sup.5 cells/mL. When the cell density reached about 80 to 85%, the medium in the wells was removed. The ethanol extract of Mori Cortex Radicis (20 g/mL) prepared in Example 1-2, the hot water extract of Mori Cortex Radicis (20 g/mL) prepared in Example 1-5, the morusin (5 M) and the kuwanon G (5 M) isolated and purified in Example 2 were dissolved in DMEM (Hyclone) containing 2% HS (Hyclone), followed by its treatment to the cells to induce myotube differentiation. Meanwhile, a group treated with 0.01% DMSO instead of the sample was used as a control group. This procedure was carried out for 6 days in two days to differentiate and then dissolved in NP-40 buffer solution (ELPIS-Biotech, Daejeon, Republic of Korea) containing proteinase inhibitor cocktail. The cells dissolved in the buffer solution were transferred to a 1.5-mL tube and centrifuged at 13,000 rpm for 10 minutes to take only a supernatant. The supernatant was quantified using Bradford (Bio-Rad Laboratories Inc., Hercules, Calif., USA). The quantified proteins were boiled for 5 minutes, separated by electrophoresis on 10% SDS-PAGE, and were transferred to the nitrocellulose membrane. A p-mTOR primary antibody (Cellular signaling technology, Beverly, Mass., USA) was diluted with 2.5% bovine serum albumin (BSA) at a ratio of 1:1000 and reacted with proteins transferred to the nitrocellulose membrane for 20 hours at room temperature. The nitrocellulose membrane incubated with the diluted primary antibodies was washed three times for 10 minutes using Tris-buffer Saline Tween 20 (TBST). After washing, the anti-rabbit secondary antibodies (Bethyl Laboratories, Inc., Montgomery, Tex., USA) conjugated with horseradish peroxidase, which recognizes the primary antibody, were diluted to 1:5000 in 2.5% BSA, followed by reaction with the nitrocellulose membrane at room temperature for 2 hours and washing three times for 10 minutes using TBST. Protein bands were developed using ECL western blotting detection reagents (Amersham, Tokyo, Japan), while developed protein bands were identified using G:BOX EF imaging system (Syngene, Cambridge, UK). The results are shown in FIG. 1.

(45) As shown in FIG. 1, it was observed that the expression of p-mTOR in L6 muscle cells was increased by treatment with the ethanol extract and the hot water extract of Mori Cortex Radicis, morusin, and kuwanon G, respectively. This result verifies that the ethanol extract and the hot water extract of Mori Cortex Radicis, morusin, and kuwanon G of the present invention are excellent in increasing muscle synthesis in muscle cells.

Example 4

(46) Effect of Morusin on the Promotion of mRNA Translation in L6 Muscle Cells

(47) The experiment was carried out in the same manner as in Example 3, except that morusin prepared in Example 2-1 was used at a concentration of 0.1 and 1 M, respectively. Protein bands were identified by treating p-p70S6K and p-4EBP1 primary antibodies (Santa Cruz Biotechnology, Santa Cruz, Calif., USA), which are involved in mRNA translation, instead of p-mTOR primary antibody.

(48) As shown in FIG. 2, it was found that the expression of p-p70S6K and p-4EBP1 proteins involved in mRNA translation in L6 muscle cells was increased by treating morusin. This result confirms that morusin according to the present invention promotes the mRNA translation process for muscle synthesis in muscle cells.

Example 5

(49) Effect of Kuwanon G on the Promotion of mRNA Translation in L6 Muscle Cells

(50) Experiments were carried out in the same manner as in Example 3, except that kuwanon G prepared in Example 2-2 was used at a concentration of 1 M and 5 M, respectively. The protein band was confirmed by treating p-p70S6K and p-4EBP1 primary antibodies (Santa Cruz Biotechnology), which are involved in the mRNA translation process, instead of the p-mTOR primary antibody.

(51) As shown in FIG. 3, the expression of p-p70S6K and p-4EBP1 proteins involved in mRNA translation in L6 muscle cells was increased by treating kuwanon G. This result verifies that kuwanon G according to the present invention promotes the mRNA translation process for muscle synthesis in muscle cells.

Example 6

(52) Effect of Extract of Mori Cortex Radicis on the Promotion of mRNA Translation in L6 Muscle Cells

(53) The experiment was carried out in the same manner as in Example 3, except that the hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Mori Cortex Radicis prepared in Examples 1-2 to 1-5 were used at a concentration of 15 g/mL. The protein band was confirmed by treating p-p70S6K and p-4EBP1 primary antibodies (Santa Cruz Biotechnology), which are involved in the mRNA translation process, instead of the p-mTOR primary antibody.

(54) As shown in FIG. 4, the expression of p-p70S6K protein involved in mRNA translation in the L6 muscle cells was increased by treating the hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Mori Cortex Radicis, respectively. This result verifies that the hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Mori Cortex Radicis according to the present invention promote the mRNA translation process for muscle synthesis in muscle cells, respectively.

Example 7

(55) Effect of Morusin on Muscle Differentiation

(56) Muscle cells, L6 myoblasts (ATCC) were added to a 6-well plate at 210.sup.5 cells/ml together with DMEM (Hyclone) containing 10% FBS (Hyclone). When the cell density reached about 80 to 85%, the medium in the wells was removed. The cells were treated with morusin in Example 2-1 dissolved in DMEM (Hyclone) containing 2% HS (Hyclone) at a concentration of 0.1 and 1 M, respectively, to induce myotube differentiation. Meanwhile, a group treated with 0.01% DMSO instead of the sample was used as a control group. This procedure was carried out for 6 days in two days to differentiate and total RNA was isolated using TRIzol reagent (Invitrogen. Carlsbad, Calif., USA). The isolated total RNA was quantified using NanoDrop 1000 (Thermo Fisher Scientific Inc., MA, USA). Quantified 16 L of RNA was synthesized into cDNA using Reverse Transcriptase Premix (ELPIS-Biotech) and PCR machine (Gene Amp PCR System 2700: Applied Biosystems, MA, USA) at 42 C. for 55 minutes and 70 C. for 15 minutes. 4 L out of 16 L cDNA was amplified by PCR using the following specific primers (Bioneer, Daejeon, Republic of Korea) and PCR premix (ELPIS-Biotech). PCR was performed by repeating 30 cycles of 95 C. for 30 seconds, 60 C. for 1 minute, and 72 C. for 1 minute.

(57) TABLE-US-00001 MyoD Forwardprimer: (SEQIDNO:1) 5-TTTCGACTCACCAGACCTGC-3 Reverseprimer: (SEQIDNO:2) 5-CAGAGCCTGCAGACCTTCAA-3 Myogenin Forwardprimer: (SEQIDNO:3) 5-TTTCGCACCTGATGGACCTG-3 Reverseprimer: (SEQIDNO:4) 5-CTTTCTTGAGCCTGCGCTTC-3 -Actin: Forwardprimer: (SEQIDNO:5) 5-AGCCATGTACGTAGCCATCC-3 Reverseprimer: (SEQIDNO:6) 5-CTCTCAGCTGTGGTGCTGAA-3

(58) PCR-amplified cDNA was separated by electrophoresis on 1.5% agarose gel and cDNA band was identified using G:BOX EF imaging system (Syngene). The results are shown in FIG. 5.

(59) As shown in FIG. 5, it was observed that the expression of MyoD and myogenin mRNA was increased in L6 muscle cells by treating morusin. This result verifies that morusin according to the present invention is excellent in promoting muscle differentiation in muscle cells.

Example 8

(60) Effect of Kuwanon G on Muscle Differentiation

(61) L6 myoblast (ATCC) were cultured in the same manner as in Example 7. The cells were treated with kuwanon G, which was isolated and purified in Example 2-2 and was dissolved in DMEM (Hyclone) containing 2% HS (Hyclone), at a concentration of 1 M and 5 M, respectively, to induce myotube differentiation. Meanwhile, a group treated with 0.01% DMSO instead of the sample was used as a control group. RT-PCR was performed in the same manner as in Example 7, after proceeding this procedure for 6 days in 2 days to differentiate.

(62) As shown in FIG. 6, it was observed that the expression of MyoD and myogenin mRNA was increased in L6 muscle cells by treating kuwanon G. This result verifies that kuwanon G according to the present invention is excellent in promoting muscle differentiation in muscle cells.

Example 9

(63) Effect of Extract of Mori Cortex Radicis on Muscle Differentiation

(64) L6 myoblast (ATCC) was cultured in the same manner as in Example 7. The cells were treated with the hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Mori Cortex Radicis, which were prepared in Example 1-2 to 1-5 and were dissolved in DMEM (Hyclone) containing 2% HS (Hyclone), at a concentration of 15 g/mL, respectively, to induce myotube differentiation. Meanwhile, a group treated with 0.01% DMSO instead of the sample was used as a control group. RT-PCR was performed in the same manner as in Example 7, after proceeding this procedure for 6 days in 2 days to differentiate.

(65) As shown in FIG. 7, it was observed that the expression of MyoD and myogenin mRNA was increased in L6 muscle cells by treating the hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Mori Cortex Radicis, respectively. This result verifies that the extract of Mori Cortex Radicis according to the present invention is excellent in promoting muscle differentiation in muscle cells.

Example 10

(66) Effect of Morusin on the Inhibition of Muscle Protein Degradation

(67) L6 myoblast (ATCC) was added to a 6-well plate at 210.sup.5 cells/ml together with DMEM (Hyclone) containing 10% FBS (Hyclone). When the cell density reached about 80% to 85%, the medium in the wells was removed and myotube differentiation was induced by treating the cells with DMEM (Hyclone) containing 2% HS (Hyclone). The medium was replaced with fresh medium every two days and the differentiation was carried out for a total of 6 days. After differentiation, 0.1 M and 1 M of morusin prepared in Example 2-1 were respectively dissolved in DMEM (Hyclone) containing 50 ng/mL tumor necrosis factor alph (TNF-; PeproTech, Rocky Hills, N.J., USA), followed by their treatment on the cells. After 6 hours, total RNA was isolated using TRIzol reagent (Invitrogen). The isolated total RNA was quantified using NanoDrop 1000 (Thermo Fisher Scientific Inc.). The quantified 16 L of RNA was synthesized into cDNA using Reverse Transcriptase Premix (ELPIS-Biotech) and PCR machine (Gene Amp PCR System 2700: Applied Biosystems) at 42 C. for 55 minutes and at 70 C. for 15 minutes. The PCR was performed by repeating 30 cycles at 95 C. for 30 seconds, at 60 C. for 1 minute and at 72 C. for 1 minute with 4 l of 16 l cDNA, the following specific primers (Bioneer), and PCR premix (ELPIS-Biotech):

(68) TABLE-US-00002 Atrogin-1 Forwardprimer: (SEQIDNO:7) 5-CCCTGAGTGGCATCGCCCAA-3 Reverseprimer: (SEQIDNO:8) 5-AGGTCCCGCCCATCGCTCA-3 MuRF-1 Forwardprimer: (SEQIDNO:9) 5-GAAATGCTATGCAGAACCTG-3 Reverseprimer: (SEQIDNO:10) 5-ATTCCTGCTTGTAGATGTCG-3 -Actin: Forwardprimer: (SEQIDNO:11) 5-AGCCATGTACGTAGCCATCC-3 Reverseprimer: (SEQIDNO:12) 5-CTCTCAGCTGTGGTGCTGAA-3

(69) PCR-amplified cDNA were separated by electrophoresis on 1.5% agarose gel, and cDNA band was identified using G:BOX EF imaging system (Syngene). The results are shown in FIG. 8.

(70) As shown in FIG. 8, it was found that mRNA expression of atrogin-1 and MuRF-1 in L6 muscle cells was decreased by treating morusin. This result verifies that morusin according to the present invention is excellent in inhibiting the degradation of muscle proteins in muscle cells.

Example 11

(71) Effect of Kuwanon G on the Inhibition of Muscle Protein Degradation

(72) In the same manner as in Example 10, L6 myoblast (ATCC) were treated with DMEM (Hyclone) containing 2% HS (Hyclone) to induce microtube differentiation, followed by dissolving Kuwanon G, which was isolated and purified in Example 2-2, in DMEM (Hyclone) containing 50 ng/mL TNF- (PeproTech). Then, RT-PCR was performed in the same manner as in Example 10.

(73) As shown in FIG. 9, it was found that mRNA expression of atrogin-1 and MuRF-1 in L6 muscle cells was decreased by treating kuwanon G. This result verifies that kuwanon G according to the present invention is excellent in inhibiting the degradation of muscle proteins in muscle cells.

Example 12

(74) Effect of Extract of Mori Cortex Radicis on the Inhibition of Muscle Protein Degradation

(75) The muscle cells cultured in the same manner as in Example 10 were treated with DMEM (Hyclone) containing 2% HS (Hyclone) to induce myotube differentiation. The hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Mori Cortex Radicis prepared in Examples 1-2 to 1-5 was dissolved in the DMEM (Hyclone) containing 50 ng/mL TNF- (PeproTech) at a concentration of 10 g/mL, respectively, and then RT-PCR was performed in the same manner as in Example 10.

(76) As shown in FIG. 10, it was found that mRNA expression of atrogin-1 and MuRF-1 in L6 muscle cells was decreased by treating the hexane extract, the ethyl acetate extract, the ethanol extract, and the hot water extract of Mori Cortex Radicis, respectively. This result verifies that the extract of Mori Cortex Radicis according to the present invention is excellent in inhibiting the degradation of muscle proteins in muscle cells.

Example 13

(77) Effect of High Pressure Extracts of Mori Cortex Radicis on Muscle Formation

(78) The muscle cells were treated with the ultra-high pressure extract of Mori Cortex Radicis prepared in Example 1-6, the supercritical fluid extract of Mori Cortex Radicis prepared in Example 1-7, and the subcritical fluid extract of Mori Cortex Radicis prepared in Example 1-8, respectively, at a concentration of 20 ppm, in the same manner as in Example 3. The p-mTOR protein band was developed using ECL Western Blotting Detection Reagents (Amersham, Tokyo, Japan) and a density of the developed protein band was measured using G:BOX EF imaging system (Syngene, Cambridge, UK). The relative density of the protein bands of the experimental group treated with the sample was expressed as a percentage (%), while the density of the control protein band was designated as 100%.

(79) The results are shown in Table 1 below.

(80) TABLE-US-00003 TABLE 1 Effect of high pressure extracts of Mori Cortex Radicis on the increase of the expression level of p-mTOR protein Experimental group Relative density (%) Control group 100 Example 1-6 142 Example 1-7 131 Example 1-8 136

(81) As shown in Table 1, it was confirmed that the ultra-high pressure extract, the supercritical fluid extract, and the subcritical fluid extract of Mori Cortex Radicis increase the protein expression of the major gene p-mTOR which is involved in the improvement of muscular function.

Example 14

(82) Evaluation on the Effect of Increasing Muscle Mass in Animal Models

(83) Five-week-old Wistar rats were adapted for 1 week and 100 ng/g of TNF- was supplied for 2 weeks to induce muscular atrophy. Then, groups were randomly assigned on the basis of body weight and divided into five groups of 8 rats per each group. 500 mg/kg body weight of the ethanol extract of Mori Cortex Radicis prepared in Example 1-2, 500 mg/kg body weight of the hot-water extract of Mori Cortex Radicis prepared in Example 1-5, 300 mg/kg body weight of the morusin prepared in Example 2-1, and 300 mg/kg body weight of the kuwanon G prepared in Example 2-2 were suspended in 0.25% carboxymethylcellulose, respectively, followed by their administration on experimental groups once a day for 8 weeks at regular time. A control group was administered with TNF- suspended in the same amount of 0.25% carboxymethyl cellulose ingested by the experimental groups.

(84) After 8 weeks of administration, the muscles under the right calf were excised and weighed with microbalance (Mettler PE 160, USA). As a result, as shown in Table 2, the weight of muscle was significantly (p<0.01) increased by 20.78%, 17.35%, 23.97%, and 22.60% in the groups administered with the ethanol extract and the hot-water extract of Mori Cortex Radicis, morusin, and kuwanon G, respectively. These results confirm that the extracts of Mori Cortex Radicis, and morusin & kuwanon G isolated from Mori Cortex Radicis according to the present invention are effective for increasing muscle mass.

(85) TABLE-US-00004 TABLE 2 Weight of calf muscle per treated material Average calf muscle weight Experimental groups (mg) Control group 438 20.8 Ethanol extract of Mori Cortex Radicis 529 30.5 Hot water extract of Mori Cortex Radicis 514 19.5 Morusin 543 32.9 Kuwanon G 537 15.2

(86) Hereinafter, there are disclosed preparation examples for preparing a pharmaceutical composition, a food composition, or a cosmetic composition, the compositions comprising, as an active ingredient, morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples 1 & 2. However, these preparation examples are not intended to limit the present invention by any means, but merely to be illustrative. The pharmaceutical, food or cosmetic compositions of Preparation Examples 1 to 3, each of which comprises morusin, kuwanon G, or the extract of Mori Cortex Radicis and is excellent in preventing or treating muscle diseases or improving muscular function, were prepared according to the conventional methods using the following components and composition ratios.

Medicine

(87) <1-1> Powder

(88) 50 mg of morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples 1 and 2, and 2 g of crystalline cellulose were mixed thoroughly and then, filled and sealed in a sealed package to obtain a powder preparation.

(89) <1-2> Tablets

(90) 50 mg of morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples 1 and 2, 400 mg of crystalline cellulose, and 5 mg of magnesium stearate were mixed thoroughly and then formulated according to conventional methods to obtain a tablet preparation.

(91) <1-3> Capsules

(92) 30 mg of morusin, kuwanon G. or the extract of Mori Cortex Radicis of Examples 1 and 2, 100 mg of whey protein, 400 mg of crystalline cellulose, and 6 mg of magnesium stearate were mixed thoroughly and then filled in a gelatin capsule according to conventional methods to obtain a capsule preparation.

(93) <1-4> Injection

(94) According to the conventional injection preparation method, the active ingredient was dissolved in distilled water for injection and the pH was adjusted to about 7.5. Subsequently, 100 mg of morusin or kuwanon G of Example 2, distilled water for injection, and pH adjusting agent were mixed. The mixture was filled in a 2 mL ampule and sterilized to prepare an injection.

Food

(95) <2-1> Preparation of Health Food

(96) 1000 mg of morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples 1 and 2, 70 g of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 g of vitamin B12, 10 mg of vitamin C, 10 g of biotin, 1.7 mg of Nicotinic amide, 50 g of folic acid, 0.5 mg of calcium pantothenate, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium monophosphate, 55 mg of dibasic calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride may be mixed to prepare a health food. The composition ratio may be arbitrarily changed. According to general health food manufacturing methods, after mixing the above components, granules are prepared for use in the manufacture of a health food composition according to a conventional method.

(97) <2-2> Preparation of Health Drinks

(98) According to general health drink manufacturing methods, 1000 mg of morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples land 2, 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of plum concentrate and 1 g of taurine, and purified water were mixed to make 900 ml in total. After stirring for 1 hour at 85 C., the resulting solution is was filtered and placed in a sterilized 2 L container, sealed, sterilized and refrigerated for use in the manufacture of a health beverage composition.

(99) <2-3> Chewing Gum

(100) 20 wt % of gum base, 76.9 wt % of sugar, 1 wt % of flavor, 2 wt % of water and 0.1 wt % of morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples 1 and 2 were mixed to be prepared into a chewing gum according to well-known methods.

(101) <2-4> Candy

(102) 60 wt % of sugar, 39.8 wt % of starch syrup, 0.1 wt % of flavor and 0.1 wt % of morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples 1 and 2 were mixed to be prepared into candy according to well-known methods.

(103) <2-5> Biscuit

(104) 25.59 wt % of first grade soft flour, 22.22 wt % of first grade plain flour, 4.80 wt % of refined sugar, 0.73 wt % of salt, 0.78 wt % of glucose, 11.78 wt % of palm shortening, 1.54 wt % of ammonium, 0.17 wt % of sodium bicarbonate, 0.16 wt % of sodium sulfite, 1.45 wt % rice powder, 0.0001 wt % of vitamin B, 0.04 wt % of milk flavor, 20.6998 wt % of water, 1.16 wt % of whole milk powder, 0.29 wt % of alternative milk powder, 0.03 wt % of calcium phosphate 1, 0.29 wt % of sulfuric salt, 7.27 wt % of spray milk, and 1 wt % of morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples 1 and 2 were mixed to be prepared into biscuit according to well-known methods.

Cosmetics

(105) <3-1> Nourishing Lotion (Milk Lotion)

(106) According to conventional methods, nourishing lotion was prepared with morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples 1 and 2 and the following components at a ratio as shown in Table 3 below.

(107) TABLE-US-00005 TABLE 3 Composition ingredients Preparation Example 3-1 (wt %) Morusin, kuwanon G, or 2.0 the extract of Mori Cortex Radicis Squalane 5.0 Wax 4.0 Polysorbate 60 1.5 Sorbitan sesquioleate 1.5 Liquid paraffin 0.5 Caprylic/capric triglyceride 5.0 Glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Triethanolamine 0.2 Preservatives, coloring agent, q.s. fragrance Purified water To 100

(108) <3-2> Softening Lotion (Skin Lotion)

(109) According to conventional methods, skin lotion was prepared with morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples 1 and 2 and the following components at a ratio as shown in Table 4 below.

(110) TABLE-US-00006 TABLE 4 Composition ingredients Preparation Example 3-2 (wt %) Morusin, kuwanon G, or 2.0 The extract of Mori Cortex Radicis Glycerin 3.0 Butylene glycol 2.0 Propylene glycol 2.0 Carboxyvinyl polymer 0.1 PEG 12 Nonyl phenyl ether 0.2 Polysorbate 80 0.4 Ethanol 10.0 Triethanolamine 0.1 Preservatives, coloring agent, q.s. fragrance Purified water To 100

(111) <3-3> Nourishing Cream

(112) According to conventional methods, nutritional cream was prepared with morusin, kuwanon G, or the extract of Mori Cortex Radicis of Example 1 and 2 and the following components at a ratio as shown in Table 5 below.

(113) TABLE-US-00007 TABLE 5 Compounding ingredient Preparation Example 3-3 (wt %) Morusin, kuwanon G, or 2.0 the extract of Mori Cortex Radicis Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 PEG60 Hardened castor oil 2.0 Liquid paraffin 10 Squalane 5.0 Caprylic/capric triglyceride 5.0 Glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservatives q.s. Coloring agent q.s. Fragrance q.s. Purified water To 100

(114) <3-4> Massage Cream

(115) According to conventional methods, massage cream was prepared with morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples 1 and 2 and the following components at a ratio as shown in Table 6 below.

(116) TABLE-US-00008 TABLE 6 Composition ingredients Preparation Example 3-4 (wt %) Morusin, kuwanon G, or 1.0 the extract of Mori Cortex Radicis Wax 10.0 Polysorbate 60 1.5 PEG60 Hardened castor oil 2.0 Sorbitan sesquioleate 0.8 Liquid paraffin 40.0 Squalane 5.0 Caprylic/capric triglyceride 4.0 Glycerin 5.0 Butylene glycol 3.0 Propylene glycol 3.0 Triethanolamine 0.2 Preservatives, coloring agent, q.s. fragrance Purified water To 100

(117) <3-5> Pack

(118) According to conventional methods, a pack was prepared with morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples 1 and 2 and the following components at a ratio as shown in Table 7 below.

(119) TABLE-US-00009 TABLE 7 Composition ingredients Preparation Example 3-5 (wt %) Morusin, kuwanon G, or 1.0 the extract of Mori Cortex Radicis Polyvinyl alcohol 13.0 Sodium carboxymethylcellulose 0.2 Glycerin 5.0 Allantoin 0.1 Ethanol 6.0 PEG12 nonyl phenyl ether 0.3 Polysorbate 60 0.3 Preservatives, coloring agent, q.s. fragrance Purified water To 100

(120) <3-6> Gel

(121) According to conventional methods, a gel was prepared with morusin, kuwanon G, or the extract of Mori Cortex Radicis of Examples 1 and 2 and the following components at a ratio as shown in Table 8 below.

(122) TABLE-US-00010 TABLE 8 Composition ingredients Preparation Example 3-6 (wt %) Morusin, kuwanon G, or 0.5 the extract of Mori Cortex Radicis Ethylenediamine sodium acetate 0.05 Glycerin 5.0 Carboxyvinyl polymer 0.3 Ethanol 5.0 PEG60 Hardened castor oil 0.5 Triethanolamine 0.3 Preservatives, coloring agent, q.s. fragrance Purified water To 100