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

Abstract

The purpose of the present invention is to provide a muscle repair / strengthening promoter that is more effective than conventional promoters. The muscle fiber differentiation promoter contains a component contained in an edible bird's nest as an active ingredient, and promotes differentiation of cells into muscle fibers. In particular, by extracting the active ingredient from the edible bird's nest through hydrolysis treatment, the active ingredient can be better extracted without causing any damage to the active ingredient contained in the edible bird's nest as compared with conventional methods in which the active ingredient is extracted through heat treatment. Accordingly, the effect of promoting muscle repair / strengthening by the active ingredient contained in the edible bird's nest can be enhanced.

Description

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

[0001] The present invention relates to a myofiber differentiation promoter and a food composition for promoting myofiber differentiation.

[0002] Muscle injury and repair are important physiological processes triggered by various factors, including exercise, trauma, and aging. Muscle injury is accompanied by the destruction of muscle fibers, activation of an inflammatory response, and the initiation of repair processes, including activation and differentiation of muscle satellite cells, remodeling of the extracellular matrix, and formation of new muscle fibers (Non-Patent Document 1).

[0003] In recent years, there has been an increase in research into the effects of nutritional supplements and specific food components on muscle repair (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, activation of muscle satellite cells, alleviation of oxidative stress, and regeneration of muscle fibers (Non-Patent Document 4).

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

[0005] It is also known that sialic acid has an improving effect on sarcopenia, a condition in which muscle mass decreases with age, leading to a decline in muscle strength and physical function (Patent Document 1). Furthermore, it is known that sialic acid derived from swallow's nest activates cell growth factors (Patent Document 2). It is also known that oral administration of sialic acid to a mouse model improved the contractile force of skeletal muscle (Non-Patent Document 5).

[0006] JP 2018-030813 A JP 2009-234980 A

[0007] Brack AS, Rando TA. Tissue-specific stem cells: lessons from the skeletal muscle satellite cell. Cell Stem Cell. 2012;10(5):504-514.Phillips SM, Van Loon LJ. Dietary protein for athletes: from requirements to optimal adaptation. J Sports Sci. 2011;29 Suppl 1: S29-S38.Cannon JG, Orencole SF, Fielding RA, et al. Acute phase response in exercise: interaction of age and vitamin E on neutrophils and muscle enzyme release. Am J Physiol. 1990;259(6 Pt 2): R1214-R1219.Powers SK, Jackson MJ. Exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production. Physiol Rev. 2008;88(4):1243-1276.Diego Moreno-Perez et al. Clinical Neurology, 2009, Vol.49, No.11, pp.852-855.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.

[0008] However, there have been concerns that animal proteins and amino acids extracted from milk components such as whey protein can disrupt the balance of the intestinal flora, leading to poor physical condition, rough skin, and an impact on the immune system (Non-Patent Document 6).

[0009] Therefore, an object of the present invention is to provide a muscle repair enhancement promoter and the like that can suppress adverse effects on physical condition.

[0010] A first aspect of the present invention is a muscle repair enhancement promoter that promotes muscle repair or muscle building, and is a muscle repair enhancement promoter that contains a component contained in bird's nest as an active ingredient.

[0011] A second aspect of the present invention is the muscle repair enhancement promoter of the first aspect, wherein the active ingredient is a component contained in an aqueous solution obtained by hydrolyzing bird's nest without undergoing an extraction treatment.

[0012] A third aspect of the present invention is the muscle repair enhancement promoter of the second aspect, wherein the hydrolysis is carried out so that the proportion of bird's nest-derived products having a molecular weight of 6000 or less is in the range of 40-60%.

[0013] A fourth aspect of the present invention is the muscle repair enhancing promoter of the second aspect, wherein the hydrolysis is carried out by preparing a solution so that the weight percentage of bird's nest is in the range of 5-15 ww%.

[0014] A fifth aspect of the present invention is the muscle repair enhancement promoter according to any one of the first to fourth aspects, which promotes muscle repair or enhancement by promoting differentiation of cells into muscle fibers.

[0015] A sixth aspect of the present invention is a food composition for promoting muscle repair and enhancement, which promotes muscle repair or enhancement, and which contains a component contained in swallow's nest as an active ingredient.

[0016] The present invention may be considered as a myofiber differentiation promoter that promotes the differentiation of cells into muscle fibers and contains a component contained in bird's nest as an active ingredient.The present invention may also be considered as a food composition for promoting myofiber differentiation that contains a component contained in bird's nest as an active ingredient and promotes the differentiation of cells into muscle fibers.

[0017] It has not been known in the past that the active ingredients contained in bird's nest have any effect on repairing or strengthening muscles. However, the present inventors have verified and discovered that the ingredients contained in bird's nest have such effects in each aspect of the present invention.

[0018] 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 enhancer that can maintain or improve the environment of the intestinal flora.

[0019] Furthermore, according to the second aspect of the present invention, it is possible to obtain the active ingredient without undergoing an extraction process that may cause some of the active ingredient to escape from the bird's nest.

[0020] 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 ingredient contained in bird's nest.

[0021] Furthermore, according to a fifth aspect of the present invention, the present inventors have found that an active ingredient contained in bird's nest promotes differentiation of cells into muscle fibers.

[0022] Patent Document 1 describes that sialic acid has an improving effect on sarcopenia. However, it does not mention the effect of promoting cell differentiation into muscle fibers. Patent Document 1 only describes that sialic acid has an improving effect on sarcopenia, but it is completely unclear what specific pathway the improvement occurs through and what its effect is. Patent Document 2 and Non-Patent Document 5 also do not describe or suggest anything about promoting muscle fiber differentiation.

[0023] There are many methods for improving sarcopenia, including at least improving mitochondrial function, improving inflammation control and anabolic resistance, maintaining neuromuscular junctions (NMJs) and motor units, improving angiogenesis and capillary density (microcirculation), improving autophagy / proteostasis, nutrient assimilation (HMB / creatine, etc.), and improving endocrine secretions (testosterone, etc.). Among these, chronic inflammation is one of the main causes of sarcopenia.

[0024] As mentioned above, there are many possible methods for improving sarcopenia. Which method is effective for a particular patient depends on the symptoms of that patient. Furthermore, Patent Document 1, Patent Document 2, and Non-Patent Document 5 do not describe or suggest the specific action of swallow's nest that can be expected to improve sarcopenia.

[0025] For example, if a patient's sarcopenia is due to a deterioration in the structure or function of the neuromuscular junction (NMJ), one of the main causes, measures such as improving the patient's exercise habits, nutritional status, and neuroregulation are necessary to preserve the NMJ. Furthermore, if the performance of aging muscles declines due to sparse capillaries, angiogenesis treatments may be effective. Thus, although sarcopenia is generally referred to as a condition, there are many specific causes, and specific treatments appropriate for each cause are effective in improving it.

[0026] The myofiber differentiation promoter or food composition for promoting myofiber differentiation according to the present invention has been clearly identified as being effective for patients in need of myofiber differentiation.

[0027] The fact that the active ingredients contained in bird's nest specifically have the above-mentioned effects was discovered for the first time through research by the inventor of the present application, and is clearly different from the inventions described in Patent Documents 1 and 2 and Non-Patent Document 5.

[0028] 1 is a diagram showing the results of Western blot analysis in C2C12 cells; 2 is a diagram showing the results of measuring reactive oxygen species (ROS) in Caco-2 cells; 3 is a diagram showing the effects of bird's nest extract samples in a mouse muscle injury model, showing (a) recovery of gait, (b) increased grip strength, and (c) an increase in muscle cross-sectional area; 4 is a diagram showing the effects of bird's nest extract samples in a mouse muscle injury model, showing the suppression of gene expression of inflammatory cytokines (a) TNFα, (b) IL-6, and (c) IFNβ1.

[0029] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the following embodiments.

[0030] The inventors of the present application investigated the anti-inflammatory and muscle repair promoting effects of bird's nest hydrolyzed extract in a muscle injury model. They also examined the antioxidant effects of bird's nest hydrolyzed extract, its direct effects on muscle cells, and its indirect effects via the intestinal tract.

[0031] First, the materials and methods used in the verification will be described.

[0032] <Preparation of Bird's Nest Extract> The bird's nest extract used was prepared by M-Style Holdings, Inc., one of the applicants of the present application. The preparation procedure involved first mixing and immersing Malaysian swiftlet nests (specimens) dried to a moisture content of 10% or less with RO membrane-treated water to a specimen concentration of 5-15 wt%. Subsequently, hydrolysis was performed so that the proportion of molecular weights of 6000 or less was in the range of 40-60%.

[0033] The resulting hydrolyzed liquid was then sieved through a 100-mesh sieve to remove any residue, and then centrifuged (10,000 rpm / 20 min) to spin down the residue. The supernatant was then collected and used as the extract sample (EBN).

[0034] EBN was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was diluted with 1x PBS to prepare solutions at concentrations of 0.5, 1.0, and 1.5 mg / mL. The resulting solutions were stored at -20°C and thawed as needed.

[0035] <Preparation of H2O2 solution> H2O2 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) was adjusted to 100 mM with sterile water, sterilized by filtration using a 0.22 μm filter (Merck Millipore, Munich, Germany), and stored at -20°C. The solution was thawed as needed and used.

[0036] Next, the measurement method and analysis method will be described.

[0037] <Measurement of antioxidant activity (H-ORAC method)> The dried and crushed powder of the specimen (P) before hydrolysis and the freeze-dried powder of EBN (EBNFD) after hydrolysis were dissolved in pure water to prepare measurement samples.

[0038] The antioxidant activity of the samples was measured using the H-ORAC method, and the results were expressed as H-ORAC values ​​and calculated as Trolox equivalents.

[0039] C2C12 Cell Culture: This study used the mouse skeletal muscle-derived myoblast cell line C2C12 as a skeletal muscle cell model. C2C12 cells were subcultured in Dulbecco's Modified Eagle Medium (DMEM) (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% heat-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 in the presence of 5% CO2.

[0040] 2.0 × 10 C2C12 cells 5 Cells were seeded into 6-well plates at a concentration of 1.5 μg / mL EBN in 2% Horse Serum (HS) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 48 hours, and differentiation was induced by changing the medium to DMEM containing 2% Horse Serum (HS) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing EBN at a final concentration of 1.5 μg / mL. Controls contained an equal volume of 1x PBS as the EBN-containing medium. Subsequently, the medium was changed and EBN was added every two days for 7 days. To examine the effect of EBN on muscle injury, cells were treated with or without 200 μM H2O2 at a final concentration 24 hours before harvesting, and harvested on days 3, 5, and 7.

[0041] 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) (100x, 163-26061, FUJIFILM Wako). Protein concentration was measured using a Micro BCA Protein Assay Kit (Thermo Fisher Scientific). A total of 20-30 μg of protein was loaded onto a 10% SDS-PAGE gel. Proteins were separated by gel electrophoresis and then transferred to Amersham Hybond P PVDF 0.45 (GE Healthcare UK Ltd., Buckinghamshire, UK).

[0042] After transfer, the cells were blocked with 5% skim milk in 0.1% TBS-T for 1 hour at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C with shaking. 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), and Myosin Heavy Chain (1:1000; MAB4470, R&D Systems, Minneapolis, MN, USA).

[0043] After primary antibody incubation, HRP-conjugated anti-rabbit secondary antibody (1:2000; 7074, CST) or anti-mouse secondary antibody (1:2000; 7076, CST) was diluted in blocking buffer and incubated at room temperature for 1 hour with shaking. After secondary antibody incubation, chemiluminescence was developed using ImmunoStar Zeta (FUJIFILM Wako), and band detection was performed using LuminoGraph I (ATTO, Tokyo, Japan). Quantitative analysis of protein expression was performed using Image J software.

[0044] <Culture of Caco-2 cells> In this study, we used the human colon carcinoma-derived cell line Caco-2 as a human intestinal epithelial cell model. Caco-2 cells were subcultured in DMEM medium supplemented with 10% heat-inactivated FBS, 4 mM L-glutamine, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C in the presence of 5% CO2.

[0045] 2.0 × 10 Caco-2 cells 5 Cells were seeded into 6-well plates at a concentration of 1000 cells / well, and 24 hours later, the medium was replaced with 10% FBS-containing DMEM containing EBN at final concentrations of 0, 0.5, 1.0, or 1.5 μg / mL. Controls contained an equal volume of 1x PBS as the EBN addition. 24 hours after EBN addition, the cells were used to measure intracellular reactive oxygen species (ROS). Oxidative stress was induced by adding 200 μM H2O2 to a final concentration simultaneously with EBN.

[0046] Measurement of reactive oxygen species (ROS) in Caco-2 cells: ROS in Caco-2 cells was measured using the cell permeabilization reagent 2,7-dichlorodihydrofluorescein diacetate (DCFDA) (Abcam, Cambridge, UK). After diffusing into the cell, DCFDA is deacetylated by intracellular esterases to form a non-fluorescent compound. This compound is then oxidized by ROS to form 2',7'-dichlorofluorescein (DCF). DCF is highly fluorescent and can be detected by fluorescence spectroscopy with excitation / emission at 485 nm / 535 nm.

[0047] Caco-2 cells were incubated with DCFDA solution at a final concentration of 20 μM at 37°C for 30 minutes. After removing the DCFDA solution, the cells were suspended in 1x buffer and analyzed using a flow cytometer (CytoFlex, Beckman Coulter, Brea, CA, USA). Results were expressed as relative mean fluorescence intensity (MFI) compared to the control.

[0048] <Mouse Muscle Injury Model> C57BL / 6N mice (male, 10 weeks old) were intramuscularly injected with 200 nmol of Cardiotoxin (CTX, LATOXAN, France) in a volume of 50 μL into the left gastrocnemius muscle using a 29G microinjector. Physiological saline was also administered intramuscularly to the contralateral limb. EBN (80 mg / mouse) was orally administered immediately after CTX administration and then every 24 hours thereafter. Motor function was assessed 1–14 days after CTX administration using the Tarlov score (0: no voluntary movement; 1: barely perceptible movement; 2: leg movement but uncoordinated with the contralateral limb and no weight support; 3: alternating stepping and propulsion movements with no weight support; 4: weight support; 5: gait with mild impairment; 6: normal gait). Seven days after administration, grip strength was measured using a Bioseb grip strength meter and corrected for body weight. In addition, after fecal collection, the animals were euthanized and tissue samples were collected.

[0049] <Histological analysis of muscle tissue> The collected muscle tissue was fixed in neutral buffered formalin, embedded in paraffin, and sliced ​​at 2 μm. The tissue sections were deparaffinized, hydrated, stained with HE staining, and imaged using a KEYENCE all-in-one microscope. Muscle cross-sectional area was analyzed using the software provided with the microscope.

[0050] <Gene expression analysis of muscle tissue> RNAiso plus (Takara Bio, Japan) was added to the collected muscle and disrupted using a bead homogenizer. Chloroform was added to the disrupted extract to separate the phases. 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 isolated and reverse-transcribed to 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 intensity associated with gene amplification was measured using a LightCycler96 (Roche). Relative gene expression levels were calculated using the ΔΔCt method.

[0051] <Statistical processing> Statistical processing was performed using the Tukey HSD test, with p < 0.05 considered significant. Statistical analysis for mouse experiments was performed using GraphPad Prism 10 with multigroup comparisons using the Tukey method.

[0052] The measurement results and analysis results are described below.

[0053] <Comparison of antioxidant activity before and after hydrolysis> The antioxidant activity of the dried, ground sample (P) before hydrolysis and the freeze-dried EBN powder (EBNFD) after hydrolysis was measured using the H-ORAC method. When the Trolox-equivalent H-ORAC value per 1 g of sample was calculated, EBNFD showed a significantly higher H-ORAC value than P (Table 1).

[0054]

[0055] <Effect of EBN on the Differentiation of C2C12 Cells> In C2C12 cells to which EBN was added, the expression level of myosin heavy chain (MHC) increased and differentiation was promoted (FIGS. 1(a) and 1(b)).

[0056] <Effect of EBN on H2O2-induced oxidative stress in Caco-2 cells> The ROS level in Caco-2 cells in which oxidative stress was induced using H2O2 was reduced by the addition of EBN (Figure 2).

[0057] <Effects of EBN on a mouse muscle injury model> When EBN was administered to mice with muscle injury, recovery from muscle injury was promoted (Figure 3(a)). Grip strength in the control limb was also increased (Figure 3(b)). Histological analysis showed that EBN administration reduced the number of immature muscle fibers and increased muscle cross-sectional area (Figure 3(c)). As shown in Figure 4, gene expression analysis showed that oral administration of EBN suppressed the expression of the inflammatory cytokines (a) TNFα, (b) IL-6, and (c) IFNβ1. These findings suggest that EBN has the ability to promote muscle repair and increase muscle strength.

[0058] Based on these results, the present inventors suggested that hydrolyzed bird's nest extract (EBN) has muscle repair-promoting and anti-inflammatory effects. First, evaluation of the antioxidant activity using the H-ORAC method showed that hydrolyzed EBN exhibited significant antioxidant activity, which may contribute to the reduction of oxidative stress after muscle damage.

[0059] Furthermore, the addition of EBN was confirmed to increase MHC expression levels in promoting differentiation of C2C12 cells, indicating that EBN acts directly on muscle to promote muscle repair.

[0060] Furthermore, EBN reduced H2O2-induced oxidative stress in Caco-2 cells, suggesting that the antioxidant effect of EBN also contributes to indirect promotion of muscle repair via the intestinal tract. Further research is expected to elucidate the mechanism by which improving the intestinal environment reduces systemic inflammatory responses and promotes muscle repair.

[0061] In a mouse muscle injury model, EBN administration promoted recovery from muscle injury and increased muscle strength. In particular, the reduction in the number of immature muscle fibers, the increase in muscle cross-sectional area, and the suppression of inflammatory cytokine gene expression were observed, suggesting that EBN supports muscle repair in multiple ways. EBN's anti-inflammatory effects may suppress excessive inflammatory responses during muscle repair and promote normal repair.

[0062] These results suggest that hydrolyzed bird's nest extract (EBN) may effectively promote muscle repair and muscle strength after muscle damage through its direct effect on muscle cells and its indirect effect via intestinal cells. Based on these results, it is expected that the active ingredients contained in EBN will be used as muscle repair promoters, food compositions for muscle repair promotion, muscle fiber differentiation promoters, and food compositions for muscle fiber differentiation promotion.

Claims

1. A muscle fiber differentiation promoter, which contains an ingredient contained in swallow's nest as an active ingredient and promotes the differentiation of cells into muscle fibers.

2. A food composition for promoting muscle fiber differentiation, which contains an ingredient contained in swallow's nest as an active ingredient and promotes the differentiation of cells into muscle fibers.

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