Composition comprising aster sphathulifolius maxim extract for preventing, improving, or treating muscular diseases or for improving muscular functions

Abstract

The present invention relates to a composition comprising Aster sphathulifolius Maxim extract for preventing, improving, or treating muscular diseases and for improving muscular functions. The Aster sphathulifolius Maxim extract according to the present invention exhibits effects of inhibiting the expression of muscle protein breakdown-related factors and increasing the expression of muscle protein synthesis-related factors, as well as alleviating the reduction of muscle tissue weight and cross-sectional area of muscle fibers, and thus can be useful for preventing, improving, or treating muscular diseases and improving muscular functions.

Claims

1. A method for treating or improving muscular diseases in a subject in need thereof, the method comprising administering an effective amount of a composition comprising an Aster spathulifolius extract as an active ingredient to the subject in need thereof, wherein the extract is an extract extracted by alcohol having 1 to 6 carbon atoms, or a mixture of water and alcohol having 1 to 6 carbon atoms.

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

3. The method of claim 1, wherein the muscular disease is at least one selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle degeneration, myotonia, muscular dystrophy, amyotrophic lateral sclerosis, myasthenia, cachexia, and sarcopenia.

4. The method of claim 1, wherein the subject is a livestock selected from the group consisting of cows, pigs, chickens, ducks, goats, sheep and horses.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a result of confirming an effect of alleviating a muscle weight loss of an Aster spathulifolius extract, as a result of dividing the weight of muscle tissue by the weight of each mouse according to the administration of the Aster spathulifolius extract in a muscle atrophy mouse model.

(2) FIG. 2 illustrates a result of confirming an effect of alleviating the reduction in size of muscular fibers of an Aster spathulifolius extract, as a result of photographing H&E staining of the gastrocnemius and a result of measuring a cross-sectional area of the muscle fiber according to the administration of the Aster spathulifolius extract in the muscle atrophy mouse model.

(3) FIG. 3 illustrates a result of confirming an effect of alleviating the reduction in size of muscular fibers of an Aster spathulifolius extract, as a result of photographing H&E staining of the quadriceps and a result of measuring a cross-sectional area of the muscle fiber according to the administration of the Aster spathulifolius extract in the muscle atrophy mouse model.

(4) FIG. 4 illustrates a result of measuring changes in mRNA expression of muscle degradation-related factors in the muscle tissue according to the administration of the Aster spathulifolius extract in the muscle atrophy mouse model.

(5) FIG. 5 illustrates a result of measuring changes in expression of muscle degradation-related factors and muscle synthesis-related factors in the muscle tissue according to the administration of the Aster spathulifolius extract in the muscle atrophy mouse model.

MODE FOR CARRYING OUT INVENTION

(6) Hereinafter, a preferred embodiment will be proposed in order to help in understanding of the present invention. However, the following Examples are just provided to more easily understand the present invention and the contents of the present invention are not limited by Examples.

Example 1: Preparation of Aster spathulifolius Extract

(7) 1-1. Washing of Ground Part of Aster spathulifolius Using Water

(8) Aster spathulifolius was collected from October to December in Jeju Island, Korea, and the ground part of Aster spathulifolius was dried at room temperature to be powdered, and then the powder passed through #4 sieve was used. 10 kg of powder of the ground part of Aster spathulifolius was added with 100 L of purified water, circulatory washed at room temperature, and dehydrated, and then the washing solution was discarded. The washing and dehydrating processes were repeated twice more, and at this time, the concentration of chlorine ions in the last washing solution was 10 ppm or less.

(9) 1-2. Preparation of Ground Part Extract of Aster spathulifolius

(10) 100 L of a mixed solvent of water and ethyl alcohol having a mixing ratio (v/v) of 1:10 was added to the washed ground part powder of Aster spathulifolius, and extracted by reflux cooling at about 85° C. to 100° C. for 2 to 5 hours, and the extract was filtered through filter paper. After the extraction process was repeated twice more, the filtrate was concentrated under reduced pressure and dried to obtain 503 g of a ground part extract of Aster spathulifolius in an oily state (AE-B).

Example 2: Establishment of Muscle Atrophy-Induced Mouse Model and Measurement of Muscle Tissue Weight

(11) An animal experiment was conducted to confirm whether an Aster spathulifolius extract improved muscle atrophy in a muscle atrophy mouse model. Five-week-old male C57BL/6 mice were adapted to an animal laboratory environment for one week, and then divided into five groups according to an experimental condition, and immobilization of the legs to induce muscle atrophy and oral administration of samples were performed for two weeks. Specifically, experimental groups were divided into an untreated control group (normal group, Cont), a muscle atrophy induced group (induction group, IM), a positive control group (HMB administered group, HMB 370) induced with muscle atrophy and administered with beta-hydroxy beta-methylbutyrate (HMB) (370 mg/kg), a group (Aster spathulifolius low-dose administered group, AS 100) induced with muscle atrophy and administered with a low dose (100 mg/kg) of Aster spathulifolius extract, and a group (Aster spathulifolius high-dose administered group, AS 200) induced with muscle atrophy and administered with a high dose (200 mg/kg) of Aster spathulifolius extract. Immobilization of the legs was performed by applying a fixing tool manufactured by using a 1.5 ml microfuge tube, clips, and a Velcro tape as shown in References (Disease models & mechanisms, 8(9), 1059-1069, 2015) to one hind leg of the mouse. All samples were dissolved in 0.5% carboxymethylcellulose (CMC) and orally administered once a day for 14 days from a day of application of leg fixation. After 2 weeks of leg fixation and oral administration of the sample, the gastrocnemius and the quadriceps of one hind leg were extracted, and the weights thereof were measured, normalized in proportion to the body weight, and then compared and shown in FIG. 1. As a result, compared to the normal group (Cont), the induction group (IM) induced muscle atrophy, resulting in a weight loss of about 40% in the gastrocnemius and about 33% in the quadriceps in proportion to the body weight. In the HMB-administered group (HMB 370), as the positive control group, the gastrocnemius decreased by about 24% and the quadriceps decreased by about 17%, showing about 16% and 16% of a muscle atrophy protective ability, respectively. On the other hand, in the low-dose group of Aster spathulifolius (AS 100), the gastrocnemius decreased by about 27% and the quadriceps decreased by about 22%. In the high-dose group of Aster spathulifolius (AS 200), the gastrocnemius decreased by about 25% and the quadriceps decreased by about 18%. Accordingly, the low-dose group of Aster spathulifolius (AS 100) exhibited a muscle atrophy protective ability of about 13% and 11%, respectively, and the high-dose group of Aster spathulifolius (AS 200) exhibited a muscle atrophy protective ability of about 15% and 15%, respectively. In other words, when considering the dose, it was confirmed that the Aster spathulifolius extract (100, 200 mg/kg) greatly alleviated the muscle weight lose due to leg fixation more than hydroxyl methylbutyrate (370 mg/kg) as the positive control group (HMB 370).

Example 3: Confirmation of Effect of Alleviating Reduction in Cross-Sectional Area of Muscle Fibers by Administration of Aster spathulifolius Extract

(12) Histological analysis was conducted to confirm whether an Aster spathulifolius extract improved the reduction in cross-sectional area of muscle fibers in a muscle atrophy mouse model. The gastrocnemius and quadriceps tissues extracted in Example 2 above were fixed with 4% paraformaldehyde and then subjected to hematoxylin and eosin staining (H&E staining), and thereafter, the cross-sectional area of the dyed muscle fibers was quantified through image J software, and the results were shown in FIGS. 2 and 3.

(13) As a result, in both muscle tissues, it was confirmed that compared to the normal group (Cont), in the induction group (IM), muscle atrophy was induced to greatly reduce the muscle fibers, and in the positive control group (HMB 370), and the low dose (AS 100) and the high dose (AS 200) of Aster spathulifolius extract, the reduction in muscle fiber size was alleviated.

(14) As a result of the quantification of the cross-sectional area of each muscle fiber, in the gastrocnemius, as compared with the normal group (Cont), the induction group (IM) decreased by about 50%, and the positive control group (HMB 370), the low dose (AS 100) of Aster spathulifolius, and the high dose (AS 200) of Aster spathulifolius decreased by about 33%, 45%, and 30%, respectively. In the quadriceps, as compared with the normal group (Cont), the induction group (IM) decreased by about 47%, and the positive control group (HMB 370), the low dose (AS 100) of Aster spathulifolius, and the high dose (AS 200) of Aster spathulifolius decreased by about 28%, 45%, and 25%, respectively.

(15) In other words, in the gastrocnemius and the quadriceps, the protective ability against the cross-sectional area of muscle fibers was confirmed as 17% and 19% in the positive control group (HMB 370), 5% and 2% in the low dose (AS 100) of Aster spathulifolius, and 20% and 22% in the high dose (AS 200) of Aster spathulifolius, respectively. As a result, in the muscle atrophy mouse model, the Aster spathulifolius extract showed the protective ability against the dose-dependent reduction in the cross-sectional area of the muscle fibers, and when considering the dose, it was found to be more effective than HMB, the positive control group (HMB 370).

Example 4: Confirmation of Expression Regulation of Muscle Atrophy-Related Factors by Administration of Aster spathulifolius Extract

(16) To confirm whether the Aster spathulifolius extract improved the mRNA expression levels of muscle atrophy-related factors, muscle ring finger-1 (MuRF-1) and Atrogin-1, in a muscle atrophy mouse model, quantitative real-time PCR (qRT-PCR) was performed. The murf-1 and the atrogin-1 are representative muscle degradation-related factors that have been reported that the expression thereof is increased under a leg fixation condition with muscle specific E3 ubiquitin ligases. It has been known that the increase in murf-1 and atrogin-1 is associated with an increase in proteosome-dependent proteolysis to cause an increase in proteolysis, thereby contributing to muscle atrophy. The mRNA was extracted from the gastrocnemius extracted in Example 2 using RNA RED of Intron Co., Ltd. (Seongnam, Gyeonggi-do) according to the product instructions. The extracted mRNA was synthesized into cDNA using a cDNA synthesis kit from TaKaRa Co., Ltd. (Tokyo, Japan), and then qRT-PCR was performed using an ABI StepOnePlus™ Real-Time PCR System from Applied Biosystems Co., Ltd. (Foster City, USA).

(17) As a result, in the murf-1 gene expression results of FIG. 4, it was confirmed that the murf-1 gene expression was about 3 times higher in the induction group (IM) than the normal group (Cont). The positive control group (HMB 370), the low dose (AS 100) of Aster spathulifolius, and the high dose (AS 200) of Aster spathulifolius were confirmed to be about 1.7 times, 1.2 times, and 0.9 times, respectively. In addition, in the atrogin-1 gene expression results, it was confirmed that the atrogin-1 gene expression was about three times higher in the induction group (IM) than in the normal group (Cont). The positive control group (HMB 370), the low dose (AS 100) of Aster spathulifolius, and the high dose (AS 200) of Aster spathulifolius were confirmed to be about 1.3 times, 1.4 times, and 0.9 times, respectively.

(18) As a result, in the muscle atrophy mouse model, it was confirmed that the Aster spathulifolius extract showed the protective ability against the increase in gene expression of concentration-dependent muscle degradation-related factors, and had better efficacy than the HMB, the positive control group (HMB 370).

Example 5: Confirmation of Expression Regulation of Muscle Atrophy- and Synthesis-Related Factors by Administration of Aster spathulifolius Extract

(19) In order to confirm whether the Aster spathulifolius extract improved the protein expression levels of MuRF-1 and Atrogin-1 as muscle atrophy-related factors, and Phosphorylated Mechanistic Target of Rapamycin (p-mTOR) as a muscle synthesis-related factor in a muscle atrophy mouse model, Western blot was performed. The mTOR is an important factor involved in regulation of protein translation initiation and is a representative factor involved in regulation of protein synthesis in muscle. The mTOR is a form in which the phosphorylated form (p-mTOR) is activated, and it is known that its expression is not changed significantly under continuous immobilization conditions, but when the activation is increased, muscle atrophy is alleviated. The mTOR contributes to an increase in muscle mass by inducing muscle protein synthesis by activating two factors that initiate mRNA translation, 4E-binding protein (4EBP1) and phosphorylated 70-kDa ribosomal S6 kinase (p70S6K) (The Korea Journal of Sports Science, 20(3): 1551-1561, 2011; The International Journal of Biochemistry and Cell Biology, 43(9): 1267-1276, 2011). Atrogin-1 and MuRF-1 are factors known to contribute to muscle atrophy by increasing protein degradation as mentioned in Example 4 above. The protein was extracted from the gastrocnemius extracted in Example 2 using a lysis buffer containing cOmplete™ protease inhibitor cocktail tablets from Roche Diagnostics Co., Ltd. (Indianapolis, USA). The extracted protein was adjusted to a constant protein concentration after checking the protein concentration according to the product instructions using a Pierce™ BCA Protein Assay kit from Thermo Fisher Scientific Co., Ltd. (Rockford, USA). The same amount of protein was subjected to electrophoresis in a 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and then transferred to a polyvinylidine fluoride (PVDF) membrane using electroblotting. The membrane was blocked with 5% skim milk for 1 hour at room temperature and then incubated overnight with a primary antibody at 4° C. On the next day, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 2 hours, and then developed using a LAS3000 luminescent image analyzer of Fujifilm Co., Ltd. (Japan, Tokyo). MuRF-1, Atrogin-1, and the secondary antibody were purchased from Santa Cruz Biotechnology Co., Ltd. (Santa Cruz, USA) and the p-mTOR antibody was purchased from Cell Signaling Technology Co., Ltd. (Danvers, USA).

(20) In the p-mTOR protein expression results of FIG. 5, there was no significant difference between the normal group (Cont) and the induction group (IM), but the p-mTOR protein expression showed a large increase in the positive control group (HMB 370) and showed a dose-dependent increase even in the Aster spathulifolius extract administration groups (AS 100 and AS 200). In the results of Atrogin-1 and MuRF-1 protein expression, it was confirmed that the expression thereof was significantly increased in the induction group (IM) compared to the normal group (Cont). The Aster spathulifolius extract showed a dose-dependent decrease, and the positive control group (HMB 370) showed a similar level of expression to the low dose of Aster spathulifolius extract (AS 100).

(21) As a result, in the muscle atrophy mouse model, the Aster spathulifolius extract showed a protective ability against changes in protein expression of concentration-dependent muscle regeneration and degradation-related factors, and showed better efficacy in reducing the increase of muscle degradation-related factors.

(22) The aforementioned description of the present invention is to be exemplified, and it can be understood by those skilled in the art that the technical spirit or required features of the present invention can be easily modified in other detailed forms without changing. Therefore, it should be appreciated that the aforementioned exemplary embodiments described above are all illustrative in all aspects and are not restricted.

INDUSTRIAL APPLICABILITY

(23) As described above, the Aster spathulifolius extract according to the present invention has effects of not only inhibiting the expression of muscle degradation-related factors and increasing the expression of muscle synthesis-related factors, but also alleviating the reduction in a muscle tissue weight and a cross-sectional area of muscle fibers. Therefore, the Aster spathulifolius extract of the present invention can be not only usefully used in the prevention, improvement or treatment of muscle diseases including muscular dystrophy, but also usefully used as a composition for improving muscle functions.