Muscle fiber differentiation promoter and food composition for promoting muscle fiber differentiation

Abstract

The present application provides a muscle repair and enhancement promoting agent having a better effect than conventional ones. The present application provides a muscle fiber differentiation promoting agent containing an ingredient contained in bird's nest as an effective ingredient and promoting cell differentiation into muscle fibers. In particular, the effective ingredient is extracted by subjecting bird's nest to a hydrolysis treatment, and thus can be extracted without loss of the effective ingredient contained in bird's nest, as compared with a conventional method of extraction by heat treatment. Therefore, the effect of promoting muscle repair and enhancement by the effective ingredient contained in bird's nest can be improved.

Description

Technical Field

[0001] This invention relates to a muscle fiber differentiation promoter and a food composition for promoting muscle fiber differentiation. Background Technology

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

[0003] In recent years, research on the effects of nutritional supplements and specific food components on muscle repair has been increasing (Non-Patent Literature 2). In particular, existing literature indicates that proteins, amino acids, antioxidants, and anti-inflammatory substances have the potential to promote muscle repair (Non-Patent Literature 3). These components work by reducing inflammation, activating muscle satellite cells, alleviating oxidative stress, and promoting muscle fiber regeneration (Non-Patent Literature 4).

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

[0005] Furthermore, sialic acid is known to improve sarcopenia, a condition characterized by decreased muscle mass, strength, and physical function due to aging (Patent Document 1). Additionally, sialic acid derived from bird's nest is known to activate cell growth factors (Patent Document 2). Moreover, it is known that oral administration of sialic acid to model mice can improve skeletal muscle contractility (Non-Patent Document 5).

[0006] Prior art literature Patent documents Patent Document 1: Japanese Patent Application Publication No. 2018-030813 Patent Document 2: Japanese Patent Application Publication No. 2009-234980 Non-patent literature Non-patent literature 1: Brack AS, Rando TA. Tissue-specific stem cells: lessons from the skeletal muscle satellite cell. Cell Stem Cell. 2012;10(5):504-514. Non-patent literature 2: Phillips SM, Van Loon LJ. Dietary protein for athletes: from requirements to optimum adaptation. J Sports Sci. 2011;29 Suppl 1: S29-S38. Non-patent literature 3: Cannon JG, Orencole SF, Fielding RA, et al. Acute phaseresponse in exercise: interaction of age and vitamin E on neutrophils and muscle enzyme release. Am J Physiol. 1990;259(6 Pt 2): R1214-R1219. Non-patent literature 4: Powers SK, Jackson MJ. Exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production. Physiol Rev. 2008;88(4):1243-1276. Non-patent literature 5: Ichizo Nishino, May Christine V. Malicdan, Satoru Noguchi. Development of therapy for distal myopathy with rimmed vacuoles. Clinical Neurology. 2009;49(11):852-855. Non-patent document 6: Diego Moreno-Perez et al. Effect of a Protein Supplement on the Gut Microbiota of Endurance Athletes: A Randomized, Controlled, Double-Blind Pilot Study, Nutrients. 2018 Mar; 10(3): 337. Summary of the Invention The problem the invention aims to solve However, there are concerns that animal proteins and amino acids extracted from milk components such as whey protein may disrupt the balance of the gut microbiota, causing physical discomfort and adverse effects on skin roughness and immunity (Non-Patent Literature 6).

[0007] Therefore, the purpose of this invention is to provide a muscle repair and enhancement promoter that can inhibit adverse effects on physical condition.

[0008] Problem-solving methods A first aspect of the present invention provides a muscle repair and enhancement promoter that promotes muscle repair or enhancement, wherein the active ingredient contains components found in bird's nest.

[0009] A second aspect of the present invention provides the muscle repair and enhancement promoter described in the first aspect, wherein the active ingredient is a component contained in an aqueous solution obtained by hydrolyzing bird's nest without extraction processing.

[0010] A third aspect of the present invention provides the muscle repair and enhancement promoter described in the second aspect, wherein the hydrolysis treatment results in the proportion of components with a molecular weight of less than 6000 in the bird's nest-derived product being in the range of 40% to 60%.

[0011] A fourth aspect of the present invention provides the muscle repair and enhancement promoter described in the second aspect, wherein the hydrolysis comprises: preparing a solution with a weight percentage of bird's nest in the range of 5 to 15 ww%, and treating the solution.

[0012] The fifth aspect of the invention provides a muscle repair and enhancement promoter as described in any one of the first to fourth aspects, which promotes muscle repair or muscle enhancement by promoting cell differentiation into muscle fibers.

[0013] A sixth aspect of the present invention provides a food composition for promoting muscle repair or enhancement, wherein the active ingredient comprises components contained in bird's nest.

[0014] This invention can also be understood as a myofiber differentiation promoter that promotes cell differentiation into myofiber, and its active ingredient contains components found in bird's nest. Furthermore, this invention can also be understood as a food composition for promoting myofiber differentiation, which, as an active ingredient, contains components found in bird's nest and promotes cell differentiation into myofiber.

[0015] The effects of the invention Previously, it was unknown that the active ingredients in bird's nest had effects related to muscle repair or enhancement. However, in various aspects of the present invention, the inventors have discovered through verification that the ingredients contained in bird's nest have the aforementioned effects.

[0016] It is known that bird's nest can maintain or improve the intestinal flora environment. Therefore, according to various aspects of the present invention, by using bird's nest in a muscle repair enhancer, it is possible to provide a muscle repair enhancer that can maintain or improve the intestinal flora environment.

[0017] Furthermore, according to a second aspect of the present invention, effective components can be obtained without performing extraction processes that may result in the loss of certain effective components in bird's nest.

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

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

[0020] Patent Document 1 describes the ameliorative effect of sialic acid on sarcopenia. However, this document does not mention its role in promoting cell differentiation into myofibrils. Based solely on the description of its ameliorative effect on sarcopenia in Patent Document 1, it is impossible to clearly define the specific pathway by which this improvement is achieved, or its exact function. Neither Patent Document 2 nor Non-Patent Document 5 describes or implies any promotion of myofibril differentiation.

[0021] Here, at least a few methods exist for improving sarcopenia, including: improving mitochondrial function, controlling inflammation and improving anabolic resistance, maintaining the neuromuscular junction (NMJ) and motor units, promoting angiogenesis and improving capillary density (microcirculation), improving autophagy / protein homeostasis, improving nutrient anabolism (HMB, creatine, etc.), and improving endocrine substances (testosterone, etc.). Among these, chronic inflammation is one of the main factors leading to sarcopenia.

[0022] As mentioned above, various methods for improving sarcopenia can be conceived. Which method is effective for a particular patient depends on the patient's symptoms. Furthermore, neither Patent Document 1, Patent Document 2, nor Non-Patent Document 5 describes or suggests the specific effects of bird's nest on sarcopenia.

[0023] For example, when sarcopenia is caused by structural or functional deterioration of the neuromuscular junction (NMJ), a major contributing factor, measures such as improving the patient's exercise habits, nutritional status, and neuromodulation are needed to protect the NMJ. Furthermore, when the function of aging muscles declines due to capillary thinning, treatments promoting angiogenesis are considered effective. Thus, even when collectively termed sarcopenia, its specific causes are diverse, and addressing the specific causes corresponding to these diverse factors is effective in improving sarcopenia.

[0024] The myofibrous differentiation promoter or food composition for promoting myofibrous differentiation involved in this invention has been proven to be effective for patients who require myofibrous differentiation.

[0025] The specific effects achieved by the active ingredients in bird's nest are insights first discovered by the inventors of this application, and are clearly different from the inventions described in Patent Documents 1 and 2 and Non-Patent Document 5. Attached Figure Description

[0026] Figure 1 This is a graph showing the results of Western blot analysis in C2C12 cells.

[0027] Figure 2 This is a graph showing the results of reactive oxygen species (ROS) measurement in Caco-2 cells.

[0028] Figure 3 The figure shows the effect of bird's nest extract samples in a mouse muscle injury model, where (a) shows gait recovery, (b) shows increased grip strength, and (c) shows increased muscle cross-sectional area.

[0029] Figure 4 The figure shows the effect of bird's nest extract samples in a mouse muscle injury model. The figure shows that the expression of the genes encoding inflammatory cytokines (a) TNFα, (b) IFNβ1 and (c) IL-6 was inhibited. Detailed Implementation

[0030] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. It should be noted that the embodiments of the present invention are not limited to the contents described below.

[0031] Example 1 The inventors of this application investigated the anti-inflammatory effects and muscle repair-promoting effects of hydrolyzed bird's nest extract in a muscle injury model. Furthermore, the antioxidant effects, direct effects on muscle cells, and indirect effects through the intestines of the hydrolyzed bird's nest extract were verified.

[0032] First, the materials and methods used for verification will be explained.

[0033] <Preparation of Bird's Nest Extract> The bird's nest extract used was prepared by M-Style Holdings Co., Ltd., one of the applicants in this application. The preparation steps are as follows: First, using water treated with an RO membrane, Malaysian swiftlet bird's nest (sample) with a moisture content adjusted to below 10% was mixed and soaked to achieve a sample concentration in the range of 5-15 ww%. Subsequently, hydrolysis was performed to ensure that the proportion of components with a molecular weight of below 6000 was in the range of 40-60%.

[0034] The prepared hydrolysate was then passed through a 100-mesh sieve to remove residue. Further centrifugation (10,000 rpm / 20 min) was used to precipitate the residue, and the supernatant was recovered and used as the extract sample (EBN).

[0035] Centrifuge EBN at 10,000 rpm for 10 minutes, and prepare solutions with concentrations of 0.5, 1.0, and 1.5 mg / mL using 1×PBS as solvent. Store the obtained solutions at -20°C and thaw as needed before use.

[0036] <Preparation of H2O2 solution> H2O2 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was prepared to 100 mM using sterile water and then filtered and sterilized using a 0.22 μm filter membrane (Merck Millipore, Munich, Germany). The filtered and sterilized solution was stored at -20°C and thawed as needed before use.

[0037] Next, the determination and analysis methods will be explained.

[0038] <Determination of Antioxidant Activity (H-ORAC Method)> The dried pulverized powder (P) of the sample before hydrolysis and the lyophilized powder (EBNFD) after hydrolysis were dissolved in pure water and used as the test sample.

[0039] The antioxidant properties of the test samples were determined using the H-ORAC method. The results are expressed as H-ORAC values ​​and converted to Trolox equivalents.

[0040] <Culturing 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 passaged in Durbecco's Modified Eagle Medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% inactivated fetal bovine serum (FBS) (complement inactivated by heating in a 56°C incubator 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) at 37°C and 5% CO2.

[0041] With 2.0×10 5 To determine the cell / well concentration, C2C12 cells were seeded in 6-well plates. After 48 hours, the medium was replaced with DMEM containing 2% horse serum (HS) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and EBN at a final concentration of 1.5 μg / mL for differentiation induction. In the control group, 1×PBS was added at the same volume as the EBN addition. Subsequently, the medium was changed and EBN was added every two days for 7 days. To investigate the effect of EBN on muscle injury, cells were treated with or without H2O2 at a final concentration of 200 μM 24 hours prior to cell recovery, and cell recovery was performed on days 3, 5, and 7.

[0042] <Western Blot Analysis> C2C12 myotube cells were dissolved in RIPA Buffer (182-02451, FUJIFILM Wako) containing Protease Inhibitor Cocktail Set III DMSO Solution (EDTA-free) (×100, 163-26061, FUJIFILM Wako), and protein concentrations were determined using the Micro BCA Protein Assay Kit (Thermo Fisher Scientific). A total protein concentration of 20–30 μg was added to a 10% SDS-PAGE gel, and after separation by gel electrophoresis, the protein was transferred to an Amersham Hybond P PVDF 0.45 membrane (GE Healthcare UK Ltd., Buckinghamshire, UK).

[0043] After transfer to a membrane, the antibody was blocked for 1 hour at room temperature using 5% skim milk in 0.1% TBS-T solution. The antibody was then diluted with blocking buffer and incubated overnight at 4°C with shaking. The following 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 HeavyChain (1:1000; MAB4470, R&D Systems, Minneapolis, MN, USA).

[0044] Following the initial antibody reaction, 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 an ImmunoStar Zeta (FUJIFILM Wako) instrument, and band detection was performed using a Lumino GraphI (ATTO, Tokyo, Japan). Protein expression was quantitatively analyzed using ImageJ software.

[0045] <Cultivation 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 passaged in DMEM medium supplemented with 10% inactivated FBS, 4 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2.

[0046] With 2.0×10 5 To determine the cell / well concentration, Caco-2 cells were seeded in 6-well plates. After 24 hours, the medium was replaced with DMEM containing EBN at final concentrations of 0, 0.5, 1.0, and 1.5 μg / mL, respectively, and containing 10% FBS. In the control group, an equal volume of 1×PBS was added. Cells 24 hours after EBN addition were used to measure intracellular reactive oxygen species (ROS). Oxidative stress was induced by adding 200 μM H2O2 concurrently with EBN.

[0047] <Measurement of Reactive Oxygen Species (ROS) in Caco-2 Cells> The intracellular ROS levels in Caco-2 cells were measured using 2′,7′-Dichlorodihydrofluorescein diacetate (DCFDA) (Abcam, Cambridge, UK), a cell permeability assay. After diffusing into the cell, DCFDA was deacetylated by an intracellular esterase, becoming a non-fluorescent compound. Subsequently, it was oxidized by ROS to form 2′,7′-Dichlorofluorescein (DCF). DCF exhibits strong fluorescence and can be detected by fluorescence spectroscopy at excitation / emission wavelengths of 485 nm / 535 nm.

[0048] Caco-2 cells were added with a final concentration of 20 μM DCFDA solution and incubated at 37°C for 30 min. After removing the DCFDA solution, the cells were resuspended in 1×Buffer and analyzed using a flow cytometer (CytoFlex, Beckman Coulter, Brea, CA, USA). Results are expressed as mean fluorescence intensity (MFI) relative to the control group.

[0049] <Mouse Muscle Injury Model> Using a 29G Myjector syringe, 200 nmol of cardiotoxin (CTX, LATOXAN, France) was injected intramuscularly into the left gastrocnemius muscle of male C57BL / 6N mice (50 μL). Saline was injected intramuscularly into the contralateral limb. Immediately after CTX injection, EBN (80 mg / mouse) was administered orally to the mice, followed by administration every 24 hours thereafter. Motor function was evaluated using the Tarlov score on days 1–14 following CTX administration: (0: no voluntary movement; 1: barely perceptible movement; 2: foot movement, but unable to coordinate with the contralateral limbs and unable to support body weight; 3: alternating stepping and propulsive movements, unable to support body weight; 4: able to support body weight; 5: walking with mild impairment; 6: normal walking). Additionally, grip strength was measured using a bioseb dynamometer on day 7 post-administration and corrected for body weight. Furthermore, mice were euthanized after fecal collection, and tissue material was collected.

[0050] <Histological Analysis of Muscle Tissue> Muscle tissue collected and fixed in neutral buffered formalin was embedded in paraffin and then cut into 2 μm thick sections. After dewaxing and hydration, the tissue sections were stained with hematoxylin and eosin (HE) and imaged using a KEYENCE all-in-one microscope. The cross-sectional area of ​​the muscle tissue was analyzed using the microscope's accompanying software.

[0051] <Gene Expression Analysis of Muscle Tissue> RNAiso plus (Takara Bio, Japan) was added to the collected muscle tissue, and the tissue was homogenized using a bead homogenizer. Chloroform was added to the homogenized extract for phase separation. Total RNA was purified from the separated aqueous phase using the FavorPrep Tissue Total RNA Mini Kit (Favorgen, Taiwan, China). 500 ng of total RNA was reverse transcribed using ReverTra Ace qPCR RT Master Mix with gDNAremover (Toyobo Life Science) to prepare cDNA. FS Essential DNA GreenMaster (Roche) and gene-specific primers were mixed with the cDNA, and changes in fluorescence accompanying gene amplification were measured using LightCycler 96 (Roche). The relative gene expression level was calculated using the ΔΔCt method.

[0052] <Statistical Processing> Statistical analysis was performed using the Tukey HSD test, with p < 0.05 considered statistically significant. For the mouse experiments, statistical analysis was conducted using GraphPad Prism 10 and the Tukey test for multiple group comparisons.

[0053] The following is an explanation of the measurement and analysis results.

[0054] <Comparison of antioxidant effects before and after hydrolysis treatment> The antioxidant activity of the dried powder (P) before hydrolysis and the lyophilized powder (EBNFD) after hydrolysis was determined using the H-ORAC method. After calculating the Trolox equivalent H-ORAC value per 1 g of sample, it was found that EBNFD showed a very high H-ORAC value compared with P (Table 1).

[0055] [Table 1]

[0056] <Effects of EBN on C2C12 cell differentiation> In C2C12 cells supplemented with EBN, the expression level of myosin heavy chain (MHC) increased, and differentiation was promoted. Figure 1 (a) and Figure 1 (b)).

[0057] <The effect of EBN on H2O2-induced oxidative stress in Caco-2 cells> In Caco-2 cells induced by oxidative stress using H2O2, the addition of EBN resulted in a decrease in intracellular ROS levels. Figure 2 ).

[0058] <Effects of EBN in a mouse model of muscle injury> Administration of EBN to mice with muscle injury promoted the recovery of muscle injury. Figure 3 (a)). Furthermore, increased grip strength was observed in the control limb ( Figure 3 (b) Tissue analysis showed that after administration of EBN, the number of immature muscle fibers decreased and the muscle cross-sectional area increased. Figure 3 (c)). For example Figure 4 As shown, gene expression analysis revealed that oral administration of EBN inhibited the expression of the genes encoding the inflammatory cytokines (a) TNFα, (b) IFNβ1, and (c) IL-6. These results indicate that EBN promotes muscle repair and enhances muscle strength.

[0059] Based on the above results, the inventors have disclosed that hydrolyzed bird's nest extract (EBN) has the effects of promoting muscle repair and anti-inflammatory activity. First, in the evaluation of antioxidant activity using the H-ORAC method, hydrolyzed EBN showed significant antioxidant activity, which may help reduce oxidative stress after muscle damage.

[0060] Furthermore, regarding the promotion of C2C12 cell differentiation, since the addition of EBN has been confirmed to increase MHC expression, it indicates that EBN can directly act on muscle and promote muscle repair.

[0061] Furthermore, this suggests that EBN can reduce H2O2-induced oxidative stress in Caco-2 cells, therefore, the antioxidant effect of EBN may also help indirectly promote muscle repair through the gut. The mechanism by which improving the gut environment reduces systemic inflammatory responses and promotes muscle repair is expected to be further elucidated through future research.

[0062] In a mouse model of muscle injury, administration of EBN promoted muscle recovery and enhanced muscle strength. In particular, the observed reduction in the number of immature muscle fibers, increase in muscle cross-sectional area, and suppression of gene expression of inflammatory cytokines suggest that EBN can support muscle repair in multiple ways. The anti-inflammatory effect of EBN may inhibit excessive inflammatory responses during muscle repair, thereby promoting normal repair.

[0063] The above results indicate that hydrolyzed bird's nest extract (EBN) has the potential to effectively promote muscle repair and strength enhancement after injury through both direct effects on muscle cells and indirect effects through intestinal cells. Based on these results, it is expected that the active ingredients contained in EBN can be applied to muscle repair and enhancement promoters, food compositions for promoting muscle repair and enhancement, muscle fiber differentiation promoters, and food compositions for promoting muscle fiber differentiation.

Claims

1. A myofibril differentiation promoter, characterized in that: As an active ingredient, it contains components found in bird's nest and promotes cell differentiation into muscle fibers.

2. A food composition for promoting myofibril differentiation, characterized in that, As an active ingredient, it contains components found in bird's nest and promotes cell differentiation into muscle fibers.

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