Muscle repair and enhancement promoting agent and food composition for promoting muscle repair and enhancement

Abstract

The present invention aims to provide a more effective muscle repair and enhancement agent than conventional ones. [Solution] A muscle repair and strengthening agent that promotes muscle repair or strengthening, comprising components contained in bird's nest as active ingredients. In particular, by extracting the active ingredients from bird's nest via hydrolysis, the active ingredients contained in bird's nest can be extracted without damaging them, compared to conventional methods that involve heat treatment. Therefore, it is possible to enhance the muscle repair and strengthening effect of the active ingredients contained in bird's nest.

Description

Technical Field

[0001] The present invention relates to a muscle repair enhancement promoter and a food composition for muscle repair enhancement promotion.

Background Art

[0002] Muscle injury and repair are important physiological processes caused by various factors such as exercise, trauma, and aging. Muscle injury involves the destruction of muscle fibers, the activation of inflammatory responses, and the initiation of the repair process. The repair process includes the activation and differentiation of muscle satellite cells, the reconstruction of the extracellular matrix, and the formation of new muscle fibers (Non-Patent Document 1).

[0003] In recent years, research on the effects of nutritional supplements and specific food components on muscle repair has been increasing (Non-Patent Document 2). In particular, it has been shown that proteins, amino acids, antioxidants, and anti-inflammatory substances may promote muscle repair (Non-Patent Document 3). These components act through the reduction of inflammation, the activation of muscle satellite cells, the alleviation of oxidative stress, and the regeneration of muscle fibers (Non-Patent Document 4).

[0004] Among them, as effective muscle repair enhancement promoters, animal proteins and amino acids extracted from milk components such as whey protein have been considered effective.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

[0006] However, concerns have been raised that animal proteins and amino acids extracted from dairy components such as whey protein may disrupt the balance of the gut microbiota, leading to health problems, skin problems, and adverse effects on the immune system (Non-Patent Literature 5).

[0007] Therefore, the present invention aims to provide a muscle repair and enhancement agent, etc., that can suppress adverse effects on physical condition. [Means for solving the problem]

[0008] A first aspect of the present invention is a muscle repair and enhancement agent that promotes muscle repair or strengthening, the agent comprising a component contained in bird's nest as an active ingredient.

[0009] A second aspect of the present invention is a muscle repair and enhancement agent according to the first aspect, wherein the active ingredient is a component contained in an aqueous solution obtained by hydrolyzing swiftlet nest without undergoing extraction treatment.

[0010] A third aspect of the present invention is a muscle repair enhancement promoter according to the second aspect, wherein the hydrolysis is performed such that the proportion of products derived from swiftlet nests with a molecular weight of 6000 or less is in the range of 40-60%.

[0011] A fourth aspect of the present invention is a muscle repair and enhancement agent according to the second aspect, wherein the hydrolysis is carried out by preparing a solution such that the weight percentage of swiftlet nest is in the range of 5-15 ww%.

[0012] A fifth aspect of the present invention is a muscle repair and enhancement promoting agent according to any of the first to fourth aspects, which promotes muscle repair or enhancement by promoting the differentiation of cells into muscle fibers.

[0013] A sixth aspect of the present invention is a food composition for promoting muscle repair or strengthening, which contains a component contained in bird's nest as an active ingredient.

[0014] The present invention may also be considered as a muscle fiber differentiation promoter that promotes the differentiation of cells into muscle fibers, and which contains components contained in swiftlet nest as an active ingredient. [Effects of the Invention]

[0015] Conventionally, the effect of the active ingredients contained in bird's nest on muscle repair or enhancement has not been known. However, each aspect of the present invention is what the inventors verified and found that the components contained in bird's nest have such an effect.

[0016] Bird's nest is known to maintain or improve the environment of the intestinal flora. Therefore, according to each aspect of the present invention, by using bird's nest as a muscle repair enhancer, it is possible to provide a muscle repair enhancement promoter or the like that can maintain or improve the environment of the intestinal flora.

[0017] Also, according to the second aspect of the present invention, it is possible to obtain active ingredients without going through an extraction process that may let some active ingredients escape from the bird's nest.

[0018] Furthermore, according to the third and fourth aspects of the present invention, it becomes easy to produce a muscle repair enhancement promoter that effectively contains the active ingredients contained in bird's nest.

[0019] Furthermore, according to the fifth aspect of the present invention, the inventors found that the active ingredients contained in bird's nest promote the differentiation of cells into muscle fibers.

Brief Description of the Drawings

[0020] [Figure 1] It is a figure showing the result of Western blot analysis in C2C12 cells. [Figure 2] It is a figure showing the measurement result of reactive oxygen species (ROS) in Caco-2 cells. [Figure 3] It is a figure showing the action of the bird's nest extract specimen in a mouse muscle injury model, showing (a) recovery of gait, (b) enhancement of grip strength, and (c) increase in muscle cross-sectional area. [Figure 4] It is a figure showing the action of the bird's nest extract specimen in a mouse muscle injury model, showing that the expression of (a) TNFα, (b) IL-6, and (c) IFNβ1 genes, which are inflammatory cytokines, was suppressed.

Mode for Carrying Out the Invention

[0021] Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. Note that the embodiments of the present invention are not limited to the content described below.

Examples

[0022] The inventors of the present application examined the anti-inflammatory effect and muscle repair promoting effect of the hydrolyzed extract of the nest of the swiftlet (bird's nest) in a muscle disorder model. In addition, the antioxidant effect, the direct effect on muscle cells, and the indirect effect via the intestine of the hydrolyzed extract of the nest of the swiftlet were verified.

[0023] First, the materials and verification methods used for the verification will be described.

[0024] <Preparation of Swiftlet Nest Extract> The swiftlet nest extract used was prepared by EMStyle Holdings Co., Ltd. among the applicants of the present application. As the production procedure, first, the nest of the Malaysian edible-nest swiftlet (specimen) dried and adjusted to contain 10% or less moisture was mixed and immersed using water treated with an RO membrane so that the specimen concentration was in the range of 5-15 ww%. Then, hydrolysis treatment was performed so that the proportion of those having a molecular weight of 6000 or less was in the range of 40-60%.

[0025] After that, the resulting hydrolyzed solution was sieved through a 100-mesh sieve to remove residues. Further, centrifugation (10000 rpm / 20 min) was performed to sediment the residues, and the supernatant was collected as an extract specimen (EBN).

[0026] EBN was centrifuged at 10,000 rpm for 10 minutes, and a solution was prepared at concentrations of 0.5, 1.0, and 1.5 mg / mL with the supernatant using 1×PBS as a solvent. The obtained solution was stored at -20°C and thawed as appropriate when used.

[0027] <Preparation of H2O2 Solution> H2O2 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was prepared at 100 mM using sterilized water, filter sterilized with a 0.22 μm filter (Merck Millipore, Munich, Germany), stored at -20°C, and thawed as appropriate for use.

[0028] Subsequently, the measurement method and analysis method will be described.

[0029] <Measurement of Antioxidant Activity (H-ORAC Method)> The dry pulverized powder (P) of the sample before hydrolysis treatment and the lyophilized powder (EBNFD) of EBN after hydrolysis treatment dissolved in pure water were used as measurement samples.

[0030] The antioxidant property of the measurement sample was measured by the H-ORAC method. The results were expressed as H-ORAC values and calculated in terms of Trolox equivalents.

[0031] <Culture of C2C12 Cells> In this study, the mouse skeletal muscle-derived myoblast cell line C2C12 was used as a skeletal muscle cell model. C2C12 cells were subcultured at 37°C in the presence of 5% CO2 using Dulbecco's Modified Eagle Medium (DMEM) medium (Nissui pharmaceutical, Tokyo, Japan) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (complement inactivated by heating in a 56°C water bath for 30 minutes), 4 mM L-glutamine (FUJIFILM Wako), 100 U / mL penicillin (FUJIFILM Wako), and 100 μg / mL streptomycin (Meiji seika pharma, Tokyo, Japan).

[0032] C2C12 cells were seeded at 2.0×10 5Cells were seeded in 6-well plates at a concentration of cells / well, and after 48 hours, the medium was changed to DMEM medium containing 2% Horse Serum (HS) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) with a final concentration of 1.5 μg / mL of EBN, and differentiation was induced. The control was given an equal volume of 1×PBS to the amount of EBN added. The medium was then changed and EBN was added every two days for 7 days. To investigate the effect of EBN on muscle injury, cells were treated in the presence or absence of a final concentration of 200 μM H2O2 24 hours before cell harvesting, and cells were harvested on days 3, 5, and 7.

[0033] <Western blot analysis> C2C12 myotubes were lysed in RIPA Buffer (182-02451, FUJIFILM Wako) supplemented with Protease Inhibitor Cocktail Set III DMSO Solution (EDTA free) (×100, 163-26061, FUJIFILM Wako), and protein concentrations were measured using the Micro BCA Protein Assay Kit (Thermo Fisher Scientific). A total of 20-30 μg of protein was placed on a 10% SDS-PAGE gel, separated by gel electrophoresis, and then transferred to Amersham Hybond P PVDF 0.45 (GE Healthcare UK Ltd., Buckinghamshire, UK).

[0034] After transcription, blocking was performed at room temperature for 1 hour using 5% skim milk in 0.1% TBS-T. The primary antibodies were diluted with Blocking buffer and shaken overnight at 4°C. The following primary antibodies were used: β-Tublin (1:1000; 2128, Cell Signaling Technology; CST, Danvers, MA, USA), MyoD (1:5000; 18943-1-AP, Proteintech, Rosemont, IL, USA), Myosin Heavy Chain (1:1000; MAB4470, R&D Systems, Minneapolis, MN, USA).

[0035] After the primary antibody reaction, the HRP-labeled anti-Rabbit secondary antibody (1:2000; 7074, CST) or anti-Mouse secondary antibody (1:2000; 7076, CST) was diluted with Blocking buffer and shaken at room temperature for 1 hour. After the secondary antibody reaction, chemiluminescence was performed using Immunostar Zeta (FUJIFILM Wako), and band detection was performed using Lumino Graph I (ATTO, Tokyo, Japan). Quantitative analysis of protein expression was performed using Image J software.

[0036] <Culture of Caco-2 cells> In this study, the human colon cancer-derived cell line Caco-2 was used as a human intestinal epithelial cell model. Caco-2 cells were subcultured at 37°C in the presence of 5% CO2 using DMEM medium supplemented with 10% heat-inactivated FBS, 4 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0037] Caco-2 cells were seeded at 2.0×10 5Cells were seeded in 6-well plates at a density of cells / well and, after 24 hours, were replaced with DMEM medium containing 10% FBS and EBN at final concentrations of 0, 0.5, 1.0, and 1.5 μg / mL. For the control, 1×PBS equal in volume to the amount of EBN added was added. Cells 24 hours after EBN addition were used for the measurement of intracellular reactive oxygen species (ROS). Oxidative stress was induced by adding H2O2 at a final concentration of 200 μM simultaneously with EBN.

[0038] <Measurement of Reactive Oxygen Species (ROS) in Caco-2 Cells> ROS in Caco-2 cells was measured using the cell-permeable reagent 2,7-dichlorodihydrofluorescein diacetate (DCFDA) (Abcam, Cambridge, UK). DCFDA is deacetylated by intracellular esterases after diffusing into the cells to become a non-fluorescent compound. It is then oxidized by ROS to become 2',7'-dichlorofluorescein (DCF). DCF is highly fluorescent and is detected by fluorescence spectroscopy with excitation / emission at 485 nm / 535 nm.

[0039] A DCFDA solution at a final concentration of 20 μM was added to Caco-2 cells and incubated at 37°C for 30 minutes. After removing the DCFDA solution, the cells were suspended in 1×Buffer and analyzed using a flow cytometer (CytoFlex, Beckman Coulter, Brea, CA, USA). The results were expressed as relative mean fluorescence intensity (MFI) compared to the control.

[0040] <Mouse Muscle Injury Model> 200 nmol of cardiotoxin (CTX, LATOXAN, France) was administered intramuscularly to the left gastrocnemius muscle of C57BL / 6N mice (male, 10 weeks old) using a 29G Mijector in a volume of 50 μL. The opposite limb was administered intramuscularly with physiological saline. EBN (80 mg / mouse) was administered orally immediately after CTX administration, and thereafter at 24-hour intervals. Motor function was evaluated using the Tarlov score 1-14 days after CTX administration (0: no voluntary movement; 1: almost imperceptible movement; 2: leg movement but lack of coordination with the opposite limb and no weight support; 3: alternating stepping and propulsion movements with no weight support; 4: weight support present; 5: gait with mild impairment; 6: normal gait). Grip strength was measured using a dynamometer (Bioseb) 7 days after administration and corrected for body weight. In addition, tissue samples were collected after euthanasia following fecal collection.

[0041] <Histological analysis of muscle tissue> The collected muscle tissue was fixed in neutral buffered formalin, embedded in paraffin, and sectioned to 2 μm thickness. The tissue sections were deparaffinized, hydrated, and then stained with HE stain, and imaged using a KEYENCE all-in-one microscope. Muscle cross-sectional area was analyzed using the microscope's accompanying software.

[0042] <Gene expression analysis of muscle tissue> RNAiso plus (Takara Bio, Japan) was added to the collected muscle and homogenized using a bead homogenizer. Chloroform was added to the homogenate after homogenization for phase separation. Total RNA was purified from the separated aqueous layer using the FavorPrep Tissue Total RNA Mini Kit (Favorgen, Taiwan). 500 ng of total RNA was aliquoted and reverse transcribed to generate cDNA using ReverTra Ace qPCR RT Master Mix with gDNA remover (Toyobo life science). FS essential DNA Green master (Roche) and gene-specific primers were mixed with the cDNA, and the change in fluorescence associated with gene amplification was measured using a LightCycler96 (Roche). The relative expression level of the gene was calculated by the ΔΔCt method.

[0043] <Statistical analysis> Statistical analysis was performed by the Tukey HSD test, and a significant difference was defined as p < 0.05. Statistical analysis related to the mouse experiments was performed by the Tukey method for multiple-group comparison using GraphPad Prism 10.

[0044] The measurement results and analysis results are described below.

[0045] <Comparison of antioxidant effects before and after hydrolysis> The antioxidant effect was measured by the H-ORAC method for the dry pulverized powder of the sample (P) before hydrolysis treatment and the lyophilized powder of EBN (EBNFD) after hydrolysis treatment. When calculating the Trolox-equivalent H-ORAC value per 1 g of the sample, EBNFD showed a very high H-ORAC value compared to P (Table 1).

[0046]

Table 1

[0047] <Effect of EBN on the differentiation of C2C12 cells> In C2C12 cells with the addition of EBN, the expression level of Myosin Heavy Chain (MHC) increased, and differentiation was promoted (Figure 1(a) and Figure 1(b)).

[0048] <Effect of EBN on H2O2-induced oxidative stress in Caco-2 cells> In Caco-2 cells with oxidative stress induced by H2O2, the ROS level decreased with the addition of EBN (Figure 2).

[0049] <Effect of EBN in a mouse muscle injury model> When EBN was administered to muscle-injured mice, the recovery from muscle injury was promoted (Figure 3(a)). Also, the grip strength of the contralateral limb was enhanced (Figure 3(b)). Tissue analysis showed that EBN administration decreased the number of immature muscle fibers and increased the muscle cross-sectional area (Figure 3(c)). As shown in Figure 4, gene expression analysis revealed that oral administration of EBN suppressed the expression of the inflammatory cytokines (a) TNFα, (b) IL-6, and (c) IFNβ1 genes. From the above, EBN was suggested to have a muscle repair-promoting effect and a muscle strength-enhancing effect.

[0050] Based on the above results, it was suggested by the inventors that edible bird's nest hydrolysate (EBN) has a muscle repair-promoting effect and an anti-inflammatory effect. First, in the evaluation of the antioxidant effect by the H-ORAC method, EBN after hydrolysis showed significant antioxidant activity, which may contribute to reducing oxidative stress after muscle injury.

[0051] Also, in the promotion of C2C12 cell differentiation, since an increase in the MHC expression level due to the addition of EBN was confirmed, it was shown that EBN acts directly on muscle and promotes muscle repair.

[0052] Furthermore, since EBN reduced H2O2-induced oxidative stress in Caco-2 cells, it was suggested that the antioxidant effect of EBN also contributes to indirectly promoting muscle repair via the intestine. The mechanism by which improving the intestinal environment promotes the reduction of systemic inflammatory responses and muscle repair is expected to be further elucidated in future research.

[0053] In a mouse model of muscle injury, EBN administration promoted recovery from muscle injury and increased muscle strength. In particular, a reduction in the number of immature muscle fibers, an increase in muscle cross-sectional area, and suppression of inflammatory cytokine gene expression were observed, suggesting that EBN provides multifaceted support for muscle repair. The anti-inflammatory effect of EBN may suppress excessive inflammatory responses during the muscle repair process and promote normal repair.

[0054] The results above suggest that swiftlet nest hydrolysate (EBN) may effectively promote muscle repair and muscle strengthening after injury through direct action on muscle cells and indirect action via intestinal cells. Based on these results, the active ingredients contained in EBN are expected to be utilized in muscle repair and strengthening agents and food compositions for promoting muscle repair and strengthening.

Claims

1. A muscle repair and enhancement agent that promotes muscle repair or strengthening, A muscle repair and enhancement agent containing components found in bird's nest as active ingredients.

2. The muscle repair and enhancement agent according to claim 1, wherein the active ingredient is a component contained in an aqueous solution obtained by hydrolyzing swiftlet nest without undergoing extraction treatment.

3. The muscle repair and enhancement agent according to claim 2, wherein the hydrolysis is performed such that the proportion of the swallow's nest-derived products with a molecular weight of 6000 or less is in the range of 40-60%.

4. The muscle repair and enhancement agent according to claim 2, wherein the hydrolysis is carried out by preparing a solution and performing the procedure such that the weight percentage of swiftlet nest is in the range of 5-15 ww%.

5. The muscle repair and enhancement promoting agent according to claim 1, which promotes muscle repair or enhancement by promoting the differentiation of cells into muscle fibers.

6. A food composition for promoting muscle repair or enhancement, which promotes muscle repair or strengthening, A food composition for promoting muscle repair and enhancement, containing components found in bird's nest as active ingredients.

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