KR102124325B1 - COMPOSITION COMPRISING Trapa natans FOR PREVENTING, IMPROVING OR TREATING SARCOPENIA OR MUSCLE ATROPHY - Google Patents

Abstract

The present invention relates to a composition for preventing, ameliorating or treating sarcopenia or muscle atrophy, comprising a fruit extract of Trapa natans as an active ingredient. Accordingly, the present invention can prevent, ameliorate, or treat sarcopenia and muscle atrophy by preventing the reduction of myotubes related to sarcopenia and muscle atrophy, and inhibiting the expression of genes and proteins related thereto. In particular, the present invention has an advantage of having fewer side effects by using the fruit extract of Trapa natans, which is a natural extract with proven stability.

Description

Composition for prevention, improvement or treatment of muscular dystrophy or muscular dystrophy containing dry matter as an active ingredient {COMPOSITION COMPRISING Trapa natans FOR PREVENTING, IMPROVING OR TREATING SARCOPENIA OR MUSCLE ATROPHY}

The present invention relates to a composition for the prevention, amelioration, or treatment of muscular dystrophy or muscular dystrophy, which includes dry as an active ingredient, and more particularly, a food composition capable of preventing or improving muscular dystrophy or muscular dystrophy by using the fruit of dry fruits, and The present invention relates to a pharmaceutical composition capable of preventing or treating muscular dystrophy or muscular dystrophy by using the fruit of dried fruit.

Aging is a multifactorial process (especially in skeletal muscle) that is promoted by stress resistance and reduced neuromuscular function caused by cell degeneration and loss of function.

Age-related muscle atrophy and skeletal muscle mass and muscle loss are known as sakopenia. Sarcopenia is sarcopenia, which is defined as a phenomenon in which muscle fiber area or density decreases and muscle mass decreases due to a decrease in protein synthesis and an increase in protein destruction.

Sacopenia leads to muscle weakness and significantly affects physical activity and quality of life (Prior literature 1 and 2). Sacopenia is common among the elderly, and it is estimated that it occurs in 5-13% of the elderly in their 60s and 70s and 11-50% in the 80s or older (Prior Art 3).

Mechanisms of sacopenia are actively being studied, including both intrinsic and extrinsic factors such as decreased satellite cell activity in muscles, changes in hormone levels, contractile-induced damage, cell phobia, autophagy, cell death and oxidative stress. It is known to be related (Prior Art 4).

Meanwhile, glucocorticoids are known to play an important role in the development of muscular dystrophy in humans and animals. That is, it was found that muscular dystrophy in both in vivo and in vitro experiments was caused by the synthesis of glucocorticoids such as dexamethasone (hereinafter referred to as'DEX') (Prior Art 5).

In skeletal muscle, DEX is known to reduce protein synthesis and increase proteolysis through the ubiquitine-proteasome pathway (Prior Art 6). The ubiquitin-proteasome pathway has been found to mediate degradation of short-lived proteins and long-lived myofibrillar proteins (Prior literature 7).

This proteolytic system consists of three enzyme components: ubiquitin-activating E1 enzyme, the ubiquitin-conjugating E2 enzyme, and ubiquitin-ligating E3 enzyme. Ubiquitin-linked E3 enzyme plays a decisive role in identifying and targeting proteins for proteasomal degradation (Prior literature 7, Prior literature 8).

In a previous study, two muscle-specific E3 ubiquitin linking enzymes, RING-finger 1 (MuRF1) and muscle atrophy F-box (MAFbx; also known as atrogin1), were identified as markers of skeletal muscle atrophy (Prior art 9). MuRF1 and atrogin1/MAFbx expression levels are known to be induced early in the muscle atrophy process prior to muscle mass reduction (Prior Art 9). It is known that expression of atrogin1/MAFbx is regulated by forkhead box O, whereas MuRF1 transcription is regulated by nuclear factor-κB (NF-κB) (Prior Art 10).

The present inventors have completed the present invention, while researching the causes of the occurrence of muscle atrophy as described above, the fruits of the dry affect the factors involved in the process of atrophy.

Trapa natans is a small floating plant mainly found in shallow water or wetlands. It is native to Europe, Africa, and Asia, and is commonly called water chestnut in Korea. Dried fruit has been used as a tonic in traditional Chinese medicine for thousands of years, and many studies have been conducted on the therapeutic potential of dried fruit, but the pharmacological mechanism for muscle disease is not yet known.

Literature 1. Marzetti E and Leeuwenburgh C: Skeletal muscle apoptosis, sarcopenia and frailty at old age. Exp Gerontol 41: 1234 1238, 2006. Literature 2. Dupont Versteegden EE: Apoptosis in muscle atrophy: Relevance to sarcopenia. Exp Gerontol 40: 473 481, 2005. Literature 3.Artaza JN, Bhasin S, Mallidis C, Taylor W, Ma K and Gonzalez Cadavid NF: Endogenous expression and localization of myostatin and its relation to myosin heavy chain distribution in C2C12 skeletal muscle cells. J Cell Physiol 190: 170 179, 2002. Literature 4. Aoki MS, Miyabara EH, Soares AG, Saito ET and Moriscot AS: mTOR pathway inhibition attenuates skeletal muscle growth induced by stretching. Cell Tissue Res 324: 149 156, 2006. Literature 5. Glass DJ: Skeletal muscle hypertrophy and atrophy signaling pathways. Int J Biochem Cell Biol 37: 1974 1984, 2005. Literature 6. Hershko A and Ciechanover A: The ubiquitin system. Annu Rev Biochem 67: 425 479, 1998. Literature 7. Lecker SH, Jagoe RT, Gilbert A, Gomes M, Baracos V, Bailey J, Price SR, Mitch WE and Goldberg AL: Multiple types of skeletal muscle atrophy involve a common program of changes in gene expression. FASEB J 18: 39 51, 2004. Literature 8. Meng SJ and Yu LJ: Oxidative stress, molecular inflammation and sarcopenia. Int J Mol Sci 11: 1509 1526, 2010. Literature 9. Choi YH, Yamaguchi K, Oda T and Nam TJ: Chemical and mass spectrometry characterization of the red alga Pyropia yezoensis chemoprotective protein (PYP): Protective activity of the N terminal fragment of PYP1 against acetaminophen induced cell death in Chang liver cells. Int J Mol Med 35: 271 276, 2015 Literature 10. Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real time quantitative PCR and the 2( Delta Delta C(T)) method. Methods 25: 402 408, 2001.

The problem to be solved in the present invention is to provide a composition capable of suppressing the mechanism of action in which muscular dystrophy or muscular dystrophy can be suppressed, consisting of natural extracts, without burdening side effects, and effectively improving or treating diseases.

In order to solve the above problems, the present invention is to provide a composition for the prevention, improvement or treatment of muscular dystrophy or muscular dystrophy, which includes the fruit extract of dried fruit as an active ingredient.

The present invention prevents, improves or treats muscular dystrophy and muscular dystrophy by preventing the reduction of myotubes associated with muscular dystrophy and muscular dystrophy, and inhibiting the increase in the expression and protein of the gene associated with it.

In particular, the present invention has the advantage that the burden of side effects is extremely small because it uses the extract of dried fruit, which is a natural extract with proven stability.

1 is an image showing the differentiation process from C2C12 myoblasts to myotubes by date (magnification x100).
Figure 2 is a graph showing the diameter of the root canal cells in the process of differentiation into root canal cells
3 is a result of measuring the expression level of myogenin protein in the process of differentiation into root canal cells
Figure 4 is a graph showing the results of Figure 3
5 is a result of measuring the expression level of MuRF1 and MAFbx protein according to various concentrations of DEX treatment in root canal cells
Figure 6 is a graph showing the results of Figure 5
7 is an image and graph showing the diameter of the root canal cells according to DEX treatment in the root canal cells
8 is a graph showing the viability results of C2C12 muscle cells during FTN treatment
9 is an image showing the change of C2C12 root canal cells during FTN and/or DEX treatment
10 is a graph showing the results of FIG. 9
11 is a result of measuring the expression level of MuRF1 and atrogin1/MAFbx protein in C2C12 muscle cells during FTN and/or DEX treatment
12 is a graph showing the results of FIG. 11;
13 is a result of measuring the expression level of MuRF1 and atrogin1/MAFbx mRNA in C2C12 muscle cells during FTN and/or DEX treatment
14 is a graph showing the results of FIG. 13;

The terms or words used in the present specification and claims conform to the technical idea of the present invention based on the "principle that the inventor can appropriately define the concept of terms in order to best describe his or her invention" It should be interpreted as meaning and concept, not limited to ordinary or dictionary meanings.

Therefore, the embodiments shown in the embodiments and the drawings described in this specification are only the most preferred embodiments of the present invention, and do not represent all of the technical spirit of the present invention, and various equivalents can be substituted at the time of this application. It should be understood that there may be water and variations.

The fruit extract of rye according to the present invention is extracted using an organic solvent, which can be used as an extraction solvent is as follows.

Suitable polar solvents include (i) water and (ii) alcohols having 1 to 6 carbon atoms (preferably methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol), (iii) acetic acid, (iv) DMFO (Dimethyl-formamide) and (v) DMSO (Dimethyl sulfoxide).

Suitable non-polar solvents include acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1- Pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloro Methane, 1,2-dichloroethane, anneal, diethylamine, ether, carbon tetrachloride, methylene chloride, petroleum ether and THF.

The present invention is an extraction solvent, (a) water, (b) an anhydrous or lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, etc.), (c) a mixed solvent of the lower alcohol and water, (d ) It is preferable to use acetone, (e) ethyl acetate, (f) chloroform, (g) butyl acetate, (h) 1,3-butylene glycol, (i) hexane and (j) diethyl ether, etc. For easy extraction, it is more preferable to extract water, ethanol, or a mixture of water and ethanol by treating with @.

In addition, the dried fruit extract used in the present invention can be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

Example. Dry fruit extract ( Trapa natans Preparation of fructus, hereinafter referred to as'FTN'

The FTN was air-dried at room temperature and ground into powder using a mechanical grinder. About 5 g of FTN powder was added to 300 ml of 70% ethanol and then stirred at room temperature for 24 hours at 100 rpm. After filtering the final extract, the solvent was removed using a rotary vacuum evaporator (N-1200S; EYELA, Tokyo, Japan). The extract was dissolved in DMSO (dissolved in dimethyl sulfoxide, Sigma-Aldrich Chemical Co., St. Louis, MO, USA) to make a stock solution at a concentration of 25 mg/ml and stored at 4°C. This solution was diluted with a desired concentration using physiological saline before use.

Experimental Example 1. Induction of differentiation into root canal cells

1-1. Experiment preparation

C2C12 mouse skeletal muscle cells were obtained from the American Type Culture Collection (Rockville, MD, USA), 10% fetal bovine serum (FBS) and 1% penicillin in a humidification chamber containing 95% air and 5% carbon dioxide- Dulbecco's modified Eagle's medium (DMEM) supplemented with streptomycin was prepared.

1-2. Experimental process

C2C12 myoblasts were cultured to a density of 70-80% in a culture dish at a temperature of 37° C., trypsinized, and then distributed in 6-well plates (3×10 ⁴ cells/well). Dispensed cells were grown in DMEM medium with 10% FBS added at 70° C. for 24 hours at a density of 70-80%. At this time, the medium was replaced with DMEM medium containing 2% FBS to induce myotube differentiation. For reference, the medium was changed every 2 days, and differentiation was performed for 6 days.

1-3. Experiment result

As shown in Figure 1, C2C12 skeletal muscle myoblast (skeletal muscle myoblast) was induced in differentiation in a medium (mitogen-poor media) with little or no mitogen, such as 2% FBS, to form multinucleate myotubes. As shown in Figure 2, it can be seen that the cell diameter remains similar after 4, 5, and 6 days of differentiation.

Moreover, as shown in FIGS. 3 and 4, it can be seen that the expression level of myogenin, a factor that regulates end differentiation of muscle cells, was similar after 4 and 5 days and increased after 6 days. .

These results indicate that it takes 6 days to completely differentiate from myoblast to myotube.

Experimental Example 2. Measurement of protein expression through western blot 1_ Confirmation of muscle atrophy caused by DEX of root canal cells

2-1. Experiment preparation

Root canal cells were prepared as in Experimental Example 1.

2-2. Experimental process

The C2C12 root canal cells were treated with 10, 50, or 100 μM of DEX at a temperature of 37° C. for 24 hours to perform concentration screening.

Cells were recovered and treated with buffer (20 mM sucrose, 1 mM EDTA, 20 μM Tris-HCl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl ₂ and 5 μg/ml aprotinin) for 30 min. Was dissolved. Protein concentration was measured using a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). For Western blot analysis, the same amount of protein was electrophoresed on SDS (sodium dodecyl sulfate)-polyacrylamide gel, and a nitrocellulose membrane (Schellocher & Schuell, Inc., Keene, NH, USA) ). Blots were probed for 1 hour using the desired antibody and incubated with diluted enzyme-bound secondary antibody. And it was visualized by the enhanced chemiluminescent body according to the guide of the manufacturer (Amersham Pharmacia Biotech). The antibodies used are described in Table 1 and Table 2 below.

2-3. Experiment result

As shown in Figures 5 and 6, DEX treatment led to an increase in the protein expression level of MuRF1 and atrogin1/MAFbx, and the increase in the protein expression increased in a concentration-dependent manner for DEX. These data suggest that DEX can cause muscle atrophy in C2C12 root canal cells. As shown in FIG. 7, C2C12 myotubes were significantly atrophied in the 100 μM treatment group compared to the control group.

Therefore, in all the following experimental examples, experiments were performed based on a concentration of 100 μM during DEX treatment.

Experimental Example 3. Toxicity of FTN in root canal cells

3-1. Experiment preparation

Root canal cells were prepared as in Experimental Example 1.

3-2. Experimental process

For cell viability analysis, cells were incubated for 24 hours with blanks and various concentrations of FTN. Cell viability was measured by mitochondrial enzyme MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to MTT-formazan (3-(4,5-dimethylthiazol-2-yl) )-2,5-diphenyltetrazolium bromide-formazan).

3-3. Experiment result

As shown in Figure 8, cytotoxicity was not found for 24 hours after FTN treatment.

Therefore, the following Experimental Examples 4 and 5 were performed by treating FTN at a concentration of 80 μg/ml, which is the maximum concentration without toxicity, as described by Choi et al.

Experimental Example 4. Diameter measurement of root canal cells

4-1. Experiment preparation

Root canal cells were prepared as in Experimental Example 1. Concentration screening was performed by treating D2 of 10, 50, or 100 μM in C2C12 root canal cells at a temperature of 37° C. for 24 hours. C2C12 root canal cells were grouped into 4 groups; i) Serum-free medium (SFM; control cultured in DMEM containing 100 U/ml penicillin and 100 mg/ml streptomycin, ii) DEX group treated with 100 μM DEX , iii) 100 μM DEX and 80 μg /ml FTN-treated DEX+FTN group , iv) 80µg/ml FTN-treated FTN group, etc. All control and experimental groups were incubated at 37° C. for 24 hours prior to being used in the experiment.

4-2. Experimental process

2, 4, 5 and 6 days after differentiation induction, images of the root canal cells were taken using a phase contrast microscope. In addition, 100 μM concentration of DEX and/or 80 μg/ml FTN was treated for 24 hours, followed by imaging of root canal cells. A total of 50 root canal cells were measured in at least 10 random fields using ImageJ software (version 4.16; National Institutes of Health, Bethesda, MD, USA).

4-3. Experiment result

Images of the root canal cells are as shown in FIG. 9. As a result, compared to the control group, as shown in FIG. 10, the cell diameter of the DEX-treated group decreased by 52%, whereas the cell diameter of the FTN-treated group increased by 5%. In addition, the reduction in cell diameter caused by DEX was inhibited by co-treatment with FTN.

Experimental Example 5. Measurement of protein expression by western blot 2_ Effect measurement by FTN

5-1. Experiment preparation

Root canal cells were prepared as in 4-1.

5-2. Experimental process

The anti-hypertrophy effect of FTN was examined by examining the protein and mRNA expression levels of muscle atrophy markers such as MuRF1 and atrogin1/MAFbx in C2C12 myotubes treated with 100 μM DEX and 80 μg/ml FTN for 24 hours.

Cells were recovered and treated with buffer (20 mM sucrose, 1 mM EDTA, 20 μM Tris-HCl, pH 7.2, 1 mM DTT, 10 mM KCl, 1.5 mM MgCl ₂ and 5 μg/ml aprotinin) for 30 min. Was dissolved. Protein concentration was measured using a Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA). For Western blot analysis, the same amount of protein was electrophoresed on SDS (sodium dodecyl sulfate)-polyacrylamide gel, and a nitrocellulose membrane (Schellocher & Schuell, Inc., Keene, NH, USA) ). Blots were probed for 1 hour using the desired antibody and incubated with diluted enzyme-bound secondary antibody. And it was visualized by the enhanced chemiluminescent body according to the guide of the manufacturer (Amersham Pharmacia Biotech). The antibodies used are described in Table 1 and Table 2.

5-3. Experiment result

The visualized results are as shown in FIG. 11. As shown in FIG. 12, it can be seen that cells treated with DEX alone significantly increased protein levels of MuRF1 and atrogin1/MAFbx when compared to a control group in which nothing was treated.

The increase in MuRF1 and atrogin1/MAFbx proteins induced by DEX was suppressed by simultaneous treatment with 80 μg/ml FTN.

Therefore, it is thought that FTN can inhibit muscle atrophy and induce a hypertrophic pathway in C2C12 root canal cells.

Experimental Example 6. Reverse transcription polymerase chain reaction (RT-PCR)

6-1. Experiment preparation

BV2 cells were dispensed into 12-well culture plates at a density of 2x105 per well and overnight. Then, the cells were further cultured in a serum-free medium for at least 4 hours and then treated. Total RNA was isolated using TRIzol reagent ((Invitrogen, CA). Total RNA obtained from cells according to the manufacturer's guidelines to synthesize complementary DNA using M-MLV reverse transcriptase (Promega, Madison, WI). (1 µg) was used as a primer with a random hexamer.

6-2. Experimental process

PCR was performed to amplify the MuRF1 and atrogin1/MAFbx genes obtained from cDNA, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control. Oligonucleotide primers for MuRF1, atrogin1/MAFbx and GAPDH are listed in Table 3 below. RT-PCR reaction conditions were as follows; initial denaturation: 95℃ 10 minutes, 40 cycles: 95℃ 15 seconds, 55℃ 15 seconds, 72℃ 15 seconds. The amplification products obtained by PCR were separated by electrophoresis on 1% agarose gel and visualized by EtBr staining.

6-3. Experiment result

The visualized results are as shown in FIG. 13. As shown in FIG. 14, it can be seen that cells treated with DEX alone significantly increased mRNA expression levels of MuRF1 and atrogin1/MAFbx when compared to a control group in which nothing was treated.

The increase in MuRF1 and atrogin1/MAFbx mRNA induced by DEX was suppressed to the control level by simultaneous treatment with 80 μg/ml FTN.

Therefore, it is thought that FTN can inhibit muscle atrophy and induce a hypertrophic pathway in C2C12 root canal cells.

Claims (7)

Contains the extract of Trapa natans fructus as an active ingredient,
Food composition for the prevention or improvement of muscular dystrophy characterized in that the fruit extract of the dry is treated at a concentration of 80 μg/ml based on C2C12 myotube.
delete delete delete The method according to claim 1,
The muscular dystrophy is a food composition for preventing or improving muscular dystrophy, characterized by aging.
delete delete

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