COMPOSITION FOR PREVENTING AND TREATING MUSCLE DISEASE, IMPROVING MUSCLE FUNCTION OR ENHANCING MOTOR PERFORMANCE COMPRISING HYDRANGENOL OR HYDRANGEA EXTRACT AS ACTIVE INGREDIENT

Abstract

Provided is a composition for preventing and treating muscle diseases, improving muscle functions, or enhancing motor performance that comprises hydrangenol or a hydrangenol-containing extract of Hydrangea as an active ingredient. The hydrangenol or Hydrangea extract containing hydrangenol is derived from natural materials and thus can be used safely without any adverse effect, and the composition comprising the hydrangenol or Hydrangea extract can be used beneficially as a medical, food, quasi-drug, or cosmetic composition having a good effect of preventing and treating muscle diseases, improving muscle functions, and enhancing motor performance.

Claims

1. A composition for preventing and treating a muscle disease, improving a muscle function, or enhancing a motor performance, the composition comprising hydrangenol of the following Chemical Formula 1 or a Hydrangea extract containing the hydrangenol as an active ingredient: ##STR00002##

2. The composition according to claim 1, wherein the hydrangenol is isolated from the Hydrangea extract.

3. The composition according to claim 1, wherein the Hydrangea extract is obtained by extraction with at least one solvent selected from the group consisting of water, an organic solvent, or a combination of both.

4. The composition according to claim 1, wherein the hydrangenol or the Hydrangea extract is contained in an amount of 0.0001 to 100 wt. % with respect to the total weight of the composition.

5. The composition according to claim 1, wherein the composition has at least one dosage form selected from the group consisting of tablet, granule, pill, capsule, liquid, jelly, and gum, wherein the composition is for oral application.

6. The composition according to claim 1, wherein the composition has at least one dosage form selected from the group consisting of toner, essence, nourishing cream, moisturizing cream, spot, gel, lotion, ointment, patch, and aerosol, wherein the composition is for cutaneous application.

7. The composition according to claim 1, wherein the composition is a medical composition, a food composition, a quasi-drug composition, or a cosmetic composition.

8. The composition according to claim 1, wherein the muscle disease is at least one disease selected from the group consisting of muscular atrophy, muscular dystrophy, myasthenia, muscle degeneration, and sarcopenia.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0029] FIGS. 1a, 1b and 1c show the results of HPLC analyses for hydrangenol contained in Hydrangea: FIG. 1a is the HPLC spectrum of a standard substance, hydrangenol, FIG. 1b is the HPLC spectrum of Hydrangea serrata, and FIG. 1c is the HPLC spectrum of Hydrangea macrophylla.

[0030] FIG. 2 is an ESIMS (positive-ion mode) spectrum of hydrangenol.

[0031] FIG. 3 is a .sup.1H-NMR spectrum of hydrangenol.

[0032] FIG. 4 is a .sup.13C-NMR spectrum of hydrangenol.

[0033] FIG. 5 is a DEPT NMR spectrum of hydrangenol.

[0034] FIG. 6 is an HSQC NMR spectrum of hydrangenol.

[0035] FIG. 7 is an HMBC NMR spectrum of hydrangenol.

[0036] FIGS. 8a and 8b are a graph showing the muscle-related gene expression varied upon treatment with hydrangenol or a Hydrangea extract.

[0037] FIG. 9 is an image showing the muscle-related protein expression varied upon treatment with hydrangenol or a Hydrangea extract.

[0038] FIG. 10 is a graph showing the change in ATP concentration in a cell upon treatment with hydrangenol or a Hydrangea extract.

BEST MODES FOR CARRYING OUT THE INVENTION

[0039] Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to those skilled in the art that these examples are illustrative purposes only and are not construed to limit the scope of the present invention.

EXAMPLE 1

Preparation of Hydrangea Extract

[0040] The extract of Hydrangea in the composition of the present invention was prepared in the following procedures. Firstly, 20 kg of dried raw materials of Hydrangea serrata or Hydrangea macrophylla and 300 kg of purified water were mixed in an extraction tank and subjected to reflux extraction at 100° C. for 5 hours. The extracted sample was passed through a cartridge filter (10 μm), concentrated under reduced pressure and then spray-dried to yield a water-soluble powder.

EXAMPLE 2

Preparation of Hydrangenol Derived from Hydrangea Extract

[0041] The extract powder obtained in Example 1 was subjected to a gel filtration with a Diaion HP-20. Each 2 L of the mixed solutions of methanol (30%, 50%, 70%, 100%) and CH.sub.2Cl.sub.2-MeOH (1:1, v/v) was used as a developing solvent for solvent fractionation into five subfractions (392-70EDia 1˜5). The subfraction 392-70EDia4 (357.4 mg) was solvent-fractionized with Sephadex LH-20 and a developing solvent of methanol into seven subfractions (392-70EDia4a˜4g). Out of the subfractions, 392-70EDia4d was recrystallized in methanol to yield a pure amorphous compound 1 (hydrangenol).

[0042] An ESIMS (positive-ion mode) analysis was firstly conducted to identify the structure of the product obtained in Example 2, suggesting that that m/z=257[M+H].sup.+ (Refer to FIG. 2). As can be seen from the .sup.1H-NMR spectrum (Refer to FIG. 3), the methane proton (H-3) at δH 5.50 and the methylene proton (H-4) at δH 3.30 and 3.06 in strong magnetic field formed a vicinal coupling, and the chemical shift value as well as the vicinal coupling accounted for the protons being originated from the C-ring. As for the protons originated from the p-substituted benzene ring of a B-ring, the peaks H-2′ and H-3′ and the peaks H-6′ and H-5′ formed ortho couplings and showed up as a doublet (J=8.4 Hz); and the peaks H-2′ and H-6′ and the peaks H-3′ and H-5′ also formed ortho couplings and showed up as a doublet. This implicitly indicated that the product had a structure symmetric with respect to the hydroxyl group. In the 1,2,3-trisubstituted benzene of the A-ring, the protons H-5 and H-7 independently formed a coupling with the proton H-6. Hence, the protons H-5 and H-7 appeared as a doublet with the ortho couplings, and the proton H-6 showed up as a double of doublets with the meta couplings, suggesting that all the peaks corresponded to one proton.

[0043] In the .sup.13C-NMR spectrum (Refer to FIG. 4), fifteen peaks including a para-substituent appeared. It was interpreted as follows: the quaternary carbon peak at δC 172 was originated from the first carbon of the compound 1, that is, on the carbonyl group; and the peaks at δC 36.1 and δC 83.1 were originated from an aliphatic carbon and an oxygenated carbon, respectively. In addition, the DEPT NMR spectrum (Refer to FIG. 5) identified seven protonated carbons, showing that the peak at δC 36.1 was a methylene group originated from the C-4. A 2D NMR analysis was carried out to analyze the precise structures of the peaks. The precise positions of the peaks were addressed according to the HSQC (Refer to FIG. 6), and the bonding positions of substituents were determined from the HMBC (Refer to FIG. 7). That is, the peak at δH 7.26 (2H, d, J=8.4 Hz, H-2′,6′) had a correlation with the peak of C-4 at δC 36.1; whereas the peaks at δH 3.06 and δH 3.30 originated from H-4 had a correlation with the peaks at δC 83.1 (C-3), δC 119.8 (C-5), δC 110.0 (C-9), and δC 142.2 (C-10). A summary of the results and a comparison with the literatures identified the compound of Example 2 as hydrangenol (Yoshikawa M., Matsuda H., Shimoda H., Shimada H., Harada E., Naitoh Y., Miki A., Yamahara J., Murakami N. Development of Bioactive Functions in Hydrangeae Dulcis Folium. V. On the Antiallergic and Antimicrobial Principles of Hydrangeae Dulcis Folium. (2). Thunberginols C, D, and E, Thunberginol G 3′-O-Glucoside, (−)-Hydrangenol 4′-O-Glucoside, and (+)-Hydrangenol 4′-O-Glucoside. Chem. Pharm. Bull. 1996, 44: 1440-1447).

EXPERIMENTAL EXAMPLE 1

Change in Muscle-Related Gene Expression Upon Treatment with Hydrangenol or Hydrangea Extract

[0044] Each sample obtained in Examples 1 and 2 was analyzed in regards to its effects on the change in gene expression involved in the gain of muscle mass and the mitochondrial biogenesis and electron transport chain (ETC). For this experiment, C2C12 cells were differentiated for 5 days and then treated with the sample of Example 1 (25 μg/ml) or Example 2 (2.5 ug/ml) for 1 hour or 24 hours, respectively. Then, Trizol was used to extract RNA from the cell, and cDNA was synthesized from the RNA (cDNA synthesis kit, Bio-Rad). Finally, a realtime PCR (qRT-PCR) was performed (Viia7, Agilent Biosystems) using the oligomers of Table 1 as primers.

TABLE-US-00001 TABLE 1 SEQ Gene Direction Sequence (5′.fwdarw.3′) ID NO PGC-1a Forward GTCCTTCCTCCATGCCTGAC  1 Reverse GACTGCGGTTGTGTATGGGA  2 ERRa Forward GAGGTGGACCCTTTGCCTTT  3 Reverse GGCTAACACCCTATGCTGGG  4 NRF-1 Forward CTTCATGGAGGAGCACGGAG  5 Reverse ATGAGGCCGTTTCCGTTTCT  6 Tfam Forward GAGCGTGCTAAAAGCACTGG  7 Reverse CCACAGGGCTGCAATTTTCC  8 MyoD Forward CCGTGTTTCGACTCACCAGA  9 Reverse GTAGTAGGCGGTGTCGTAGC 10 Myogenin Forward GAGGAAGTCTGTGTCGGTGG 11 Reverse CCACGATGGACGTAAGGGAG 12 SDHb Forward ACTGGTGGAACGGAGACAAG 13 Reverse GTTAAGCCAATGCTCGCTTC 14 CytC Forward GGGCCTCGTTAGTGCAGCAGG 15 Reverse GGGCTCCCAGAAAAGGTTGCCT 16 COX2 Forward ACGAAATCAACAACCCCGTA 17 Reverse GGCACAACGACTCGGTTATC 18 COX4 Forward ACTACCCCTTGCCTGATGTG 19 Reverse GCCCACAACTGTCTTCCATT 20 ATP5g1 Forward TGGGGACCAGGGCAGCCATT 21 Reverse AGGGCTTGCTGCCCACACAT 22 PPAra Forward ATCCAGGGTTCAGTCCAGTG 23 Reverse GCTTAGGGACAGTGACAGGT 24 PPARd Forward GTTCCTCTGACCCTGTCCTC 25 Reverse CCAGCAAGTTTCAAGCCACT 26 UCP1 Forward CGGAAACAAGATCTCAGCCG 27 Reverse CTGACCTTCACGACCTCTGT 28 SIRT1 Forward TGCCATCATGAAGCCAGAGA 29 Reverse AACATCGCAGTCTCCAAGGA 30 GAPDH Forward AACTTTGGCATTGTGGAAGG 31 Reverse GGATGCAGGGATGATGTTCT 32

[0045] In the experimental example, the extract of Hydrangea serrata is indicated as S; the extract of Hydrangea macrophylla as M; and hydrangenol as H. “0” means a control that was treated with vehicle.

[0046] The samples of Examples 1 and 2 increase the amount of mRNA expression of peroxisome proliferator-activated receptor a (PPARα) in the skeletal muscle and promote peroxisomal β-oxidation along with PGC-1α for use as an energy source in the muscle, thereby enhancing muscle functions. In addition, the samples of Examples 1 and 2 activate PGC-1α and peroxisome proliferator-activated receptor 5 (PPARδ) along with sirtuin 1 (SIRT 1) of which expression is increased, thus enhancing absorption of glucose, oxidation of fatty acids, and mitochondrial synthesis.

[0047] MyoD is a crucial protein involved in muscle differentiation, which means that the hydrangenol of Hydrangea can accelerate the differentiation of muscle cells. Myogenin is a protein involved in skeletal muscle development as a muscle-specific transcription factor. An increase in the mRNA expression of the myogenic regulatory factor protein implies inducing the differentiation of muscle cells and the synthesis of proteins and increasing the muscle mass.

[0048] Nuclear respiratory factor 1 (NRF-1) is a transcription factor that activates the expression of proteins involved in cell growth and cellular respiration. An increase in the expression of NRF-1 results in the expression of genes regulating cellular growth, respiration, and mitochondrial DNA (mtDNA) transcription and replication. The NRF-1 is known to regulate mitochondrial transcription factor A (TFAM), which is a transcription factor of mitochondria that participates in mitochondrial genome replication and transcription. In addition, an increase in mitochondrial cytochrome C (CytC), cytochrome C oxidase subunit 2 (Cox2), and cytochrome C oxidase subunit 4 (Cox4) indicates the activation of the mitochondrial electron transport chain (ETC). A mitochondrial ATP synthase lipid-binding protein (ATG5g1) is a part of the ATP synthase of mitochondria. An increase in the gene expression of ATG5g1 results in increasing the intracellular content of ATP acting as an energy carrier in cells, which eventually boosts the activity of the cells.

[0049] As factors involved in muscle hypertrophy, tuberous sclerosis complex 1 (TSC1) and tuberous sclerosis complex 2 (TSC2) are GTPase-activating proteins (GAPs) that negatively regulate the mammalian target of rapamycin (mTOR). The mammalian target of rapamycin (mTOR) is a phosphorylated protein that controls cell growth and proliferation, cell survival rate, and protein synthesis and transcription. A decrease in the mRNA expression of TSC1 and TSC2 is activating the mTOR signaling, which increases the protein synthesis in cells and promotes the cell growth and the activity of cells, increasing the muscle cells.

[0050] It was therefore confirmed, as shown in FIG. 8, that especially, Hydrangea serrata extract S in Example 1 induced the gene expression of PGC1α, NRF-1, TFAM, MyoD, PPARα, PPARδ, ATG5g1, and SIRT1. It suggested that the absorption of glucose and fatty acids and the mitochondrial protein synthesis increased the ATP content and helped to enhance the motor performance. That was also backed up by the increased expression of SDH1b, CyctC, COX2, COX4, ATP5g1, UCP1, NRF-1, and PPARδ and the decreased expression of TSC1 and TSC2. In addition, Hydrangea macrophylla extract M increased the expression of Myogenin, COX2, ATP5g1, and PPARδ and helped to gain the muscle mass and enhance muscle functions through boosted muscle development and activation of mitochondrial electron transport chain (ETC), or the like.

[0051] The sample of Example 2, which was the effective ingredient of Example 1, regulated the amount of expression for most of genes shown in FIG. 8 and particularly promoted the expression of MyoD and Myogenin to affect the cell proliferation and the protein synthesis, thereby inducing a gain of the muscle mass.

EXPERIMENTAL EXAMPLE 2

Change in Muscle-Related Protein Expression Upon Treatment with Hydrangenol or Hydrangea Extract

[0052] Each sample obtained in Examples 1 and 2 was analyzed in regards to its effects on the change in protein expression involved in the prevention of muscle diseases, the improvement of muscle functions, and the promotion of motor performance. For this experiment, C2C12 cells were differentiated for 5 days and then treated with the sample of Example 1 (25 ug/ml) or Example 2 (2.5 ug/ml) for 1 hour or 24 hours, respectively. Then, the cell was broken down with a modified LIPA buffer, and 20 ug of each sample was used for analysis. The primary antibodies of p-ERK (9101S: Cell Signaling), p-mTOR (ab109268, Abcam), mTOR (2972, Cell Signaling), p-p70S6K (9234, Cell Signaling), p-AMPK (4186, Cell Signaling), PGC1α (ab54481, Abcam), and β-actin (A5316, Sigma) were used for the analysis.

[0053] As can be seen from FIG. 9, the samples obtained in Examples 1 and 2 increased the activity of p-AMPK and the protein expression of PGC1α. Further, in the same manner as indicated by the results of the Experimental Example 1, the absorption of glucose and fatty acids and the mitochondrial synthesis increased the ATP content and helped to enhance the motor performance. In addition, the samples of Examples 1 and activated the phosphorylation of mTOR through the phosphorylation of ERK, also known as MAPK, and the mTOR signaling followed by the phosphorylation of the subordinate protein, p70S6K, to affect the proliferation of muscle cells and the protein synthesis, inducing an increase in the muscle mass.

EXPERIMENTAL EXAMPLE 3

Change in Intracellular ATP Concentration Upon Treatment with Hydrangenol or Hydrangea Extract

[0054] The sample obtained in Example 1 was analyzed in regards to its effect on the change in intracellular ATP concentration. A mitochondrial ToxGlo™ Assay (Promega) kit was used to measure the amount of ATP in the cells treated with the sample of Example 1. The differentiated cells were treated with a 2X ATP detection reagent at the room temperature and measured in regards to the luminescence.

[0055] As can be seen from FIG. 10, the sample of Example 1 increased the amount of ATP in the cells in a concentration-dependent manner. Therefore, in the same manner as described in Experimental Examples 1 and 2, the sample of Example 1 increased the activity of mitochondria and helped to enhance the motor performance.

FORMULATION EXAMPLE 1

Preparation of Tablets

[0056] The extract of Example 1 or Hydrangenol of Example 2 was mixed with the ingredients of Table 2 or 3 and processed into tablets according to a general preparation method for tablet.

TABLE-US-00002 TABLE 2 Ingredients Unit weight (mg) Example 1 50 Corn starch 100 Lactose 100 Stearic acid 2

TABLE-US-00003 TABLE 3 Ingredients Unit weight (mg) Example 2 10 Corn starch 100 Lactose 100 Stearic acid 2

FORMULATION EXAMPLE 2

Preparation of Capsules

[0057] The extract of Example 1 or Hydrangenol of Example 2 was mixed with the ingredients of Table 4 or 5 and filled in gelatin capsules to prepare soft capsules according to a general preparation method for capsule.

TABLE-US-00004 TABLE 4 Ingredients Unit weight (mg) Example 1 50 Corn starch 100 Lactose 100 Stearic acid 2

TABLE-US-00005 TABLE 5 Ingredients Unit weight (mg) Example 2 2 Vitamin E 2.25 Vitamin C 2.25 Palm oil 0.5 Vegetable hydrogenated oil 2 Yellow lead 1 Lecithin 2.25 Filling solution for soft capsule 387.75

FORMULATION EXAMPLE 3

Preparation of Liquid

[0058] The extract of Example 1 or Hydrangenol of Example 2 was mixed with the ingredients of Table 6 or 7 and filled in a bottle or a pouch to prepare a liquid according to a general preparation method for beverage.

TABLE-US-00006 TABLE 6 Ingredients Unit weight (g) Example 1 2.5050 Xanthan gum 0.0075 Pructooligosaccharide 0.7500 Powdered coconut flower nectar 1.0500 Concentrated ssangwha-tang 1.5000 Red ginseng flavor 0.0450 Purified water 9.1425

TABLE-US-00007 TABLE 7 Ingredients Unit weight (g) Example 2  0.0205 Xanthan gum  0.0075 Pructooligosaccharide  0.7500 Powdered coconut flower nectar  1.0500 Concentrated ssangwha-tang  1.5000 Red ginseng flavor  0.0450 Purified water 20.1425

FORMULATION EXAMPLE 4

Preparation of Chewable Gel

[0059] The extract of Example 1 or Hydrangenol of Example 2 was mixed with the ingredients of Table 8 or 9 and filled in a three-sided seal pouch to prepare a chewable gel according to a general preparation method for chewable gel.

TABLE-US-00008 TABLE 8 Ingredients Unit weight (g) Example 1  2.0000 Food gel  0.3600 Carrageenan  0.0600 Calcium lactate  0.1000 Sodium citrate  0.0600 Complex scutellaria extract  0.0200 Enzymatically modified stevia  0.0440 Fructooligosaccharide  5.0000 Red grape concentrate  2.4000 Purified water 13.9560

TABLE-US-00009 TABLE 9 Ingredients Unit weight (g) Example 2  0.0200 Food gel  0.3600 Carrageenan  0.0600 Calcium lactate  0.1000 Sodium citrate  0.0600 Complex scutellaria extract  0.0200 Enzymatically modified stevia  0.0440 Fructooligosaccharide  5.0000 Red grape concentrate  2.4000 Purified water 13.9560

FORMULATION EXAMPLE 5

Preparation of Nutrient Cream

[0060] The extract of Example 1 or Hydrangenol of Example 2 was processed into the composition of Table 10 or 11 according to a general preparation method for nutrient cream.

TABLE-US-00010 TABLE 10 Ingredients Content (%) Example 1 1.0 Sitosterol 4.0 Polyglyceryl 2-oleate 3.0 3.0 Ceteareth-4 2.0 Cholesterol 3.0 Dicetyl phosphate 0.4 Concentrated glycerin 5.0 Sunflower oil 22.0 Carboxylvinyl polymer 0.5 Triethanol amine 0.5 Preservative trace Flavor trace Purified water balance

TABLE-US-00011 TABLE 11 Ingredients Content (%) Example 2 0.01 Sitosterol 4.0 Polyglyceryl 2-oleate 3.0 3.0 Ceteareth-4 2.0 Cholesterol 3.0 Dicetyl phosphate 0.4 Concentrated glycerin 5.0 Sunflower oil 22.0 Carboxylvinyl polymer 0.5 Triethanol amine 0.5 Preservative trace Flavor trace Purified water balance

[0061] The above-defined composition is given as a formulation example using a mixture of appropriate compositions. Yet the mixing ratio and the ingredients maybe varied arbitrarily under necessity.

[0062] The extract of the present invention was stable under the testing conditions for all formulation examples and hence not problematic in the stability of the dosage form.