Use of sialic acid or a composition comprising sialic acid in enhancing athletic performance or alleviating exercise-induced fatigue

Abstract

The application relates to the technical field of functional compounds, in particular to application of sialic acid or a composition containing sialic acid in enhancing sports ability or relieving sports fatigue. The composition comprises sialic acid and omega-6 fatty acid. The omega-6 fatty acid is ARA, and the mass ratio of the sialic acid to the omega-6 fatty acid is (1-20):1. The application finds that sialic acid can be used for enhancing sports ability, promoting muscle growth and relieving sports fatigue. When sialic acid is used in combination with ARA, the two components show excellent synergistic effect. The application provides a new scheme for developing food, health products or medicines for improving sports performance and relieving sports fatigue, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of functional compound technology, and more particularly to the use of sialic acid, or compositions containing sialic acid, in enhancing athletic performance or relieving exercise fatigue. Background Technology

[0002] With increasing health awareness and the rapid development of competitive sports, how to safely and effectively improve physical performance has become a core issue in sports science, nutrition, and medicine. Athletic performance not only affects athletes' competitive results but also directly impacts the exercise effects, fitness quality, and long-term health benefits of the general population. Skeletal muscle, as a vital energy metabolism organ, participates not only in motor functions during physical activity but also in physiological processes such as glucose and lipid metabolism. Its differentiation, maturation, and energy metabolism capacity directly determine the body's athletic performance. Aging, lack of exercise, and neurodegenerative diseases are all contributing factors to the loss of skeletal muscle mass. With the increasing aging of society, people are paying more attention to the benefits of exercise.

[0003] Exercise fatigue is a major physiological phenomenon limiting athletic performance, and its occurrence involves complex multi-system mechanisms. First, there is an imbalance in energy metabolism; high-intensity or prolonged exercise leads to glycogen depletion and insufficient ATP synthesis, triggering an energy crisis. Second, there is the accumulation of metabolic products; lactic acid, hydrogen ions, ammonia, and other metabolic byproducts accumulate in muscle tissue, interfering with muscle fiber contraction and causing muscle soreness and decreased strength. Third, there is oxidative stress damage; excessive free radicals are produced during exercise, attacking cell membranes, proteins, and DNA, exacerbating muscle damage and inflammatory responses. Furthermore, there is neurotransmitter imbalance; an imbalance in the ratio of neurotransmitters such as serotonin and dopamine in the central nervous system may lead to central fatigue, manifesting as insufficient motor drive and decreased attention. Finally, there is endocrine and immune regulation disorder; sustained high-intensity exercise can cause elevated cortisol levels, an imbalance in the testosterone / cortisol ratio, and temporary immunosuppression, affecting recovery ability. These fatigue mechanisms not only reduce athletic performance but may also increase the risk of sports injuries and affect the athlete's psychological state and long-term participation willingness.

[0004] Currently, commonly used methods for enhancing exercise and combating fatigue mainly include single-component nutritional supplements, synthetic drugs or hormones, and traditional dietary supplements or Chinese herbal formulas. Among these, single-component nutritional supplements (such as creatine, branched-chain amino acids, and caffeine) often target only specific mechanisms, resulting in limited effectiveness and potential side effects (such as caffeine tolerance and the impact of creatine on water metabolism). While synthetic drugs or hormones may have rapid onset of action, they often carry significant health risks and are subject to strict regulation, including doping violations. Traditional dietary supplements or Chinese herbal formulas generally lack standardized research, have unclear mechanisms of efficacy, and exhibit inconsistent absorption and utilization rates. Therefore, developing a natural or nutritional combination based on multi-target, multi-pathway synergistic effects has become an urgent need in the field of sports nutrition. Summary of the Invention

[0005] In order to address the problems existing in the prior art, the present invention provides the use of sialic acid, or a composition containing sialic acid, in enhancing athletic performance or relieving exercise fatigue.

[0006] In a first aspect, the present invention provides the use of sialic acid, or a composition containing sialic acid, in any of the following: (1) Enhance athletic ability or relieve exercise fatigue; (2) Prepare products for enhancing athletic ability or relieving exercise fatigue.

[0007] Sialic acid (SA), scientifically known as N-acetylneuraminic acid, is widely distributed in various biological tissues. It is an important component of glycoproteins, oligosaccharides, and glycolipids, typically existing as glycosides at the terminals of glycoproteins and glycolipids. It is particularly abundant in the brain, nerve tissue, blood, submandibular glands, mucins, and colostrum of mammals. Sialic acid has various physiological functions, playing a crucial role in regulating human physiological and biochemical functions, such as anti-inflammatory, antiviral, antitumor, and influenza-resistant effects. In the brain, sialic acid can promote cognitive development in infants and enhance learning and memory abilities.

[0008] Existing research has explored the treatment of GNE myopathy through 6'-sialic acid lactose supplementation, but its aim is to compensate for insufficient endogenous sialic acid synthesis and help myosin expression remain intact. No studies have yet confirmed whether sialic acid supplementation can promote myosin expression, improve muscle mass and strength, or enhance athletic performance in organisms with normal endogenous sialic acid expression. This invention, however, has found that sialic acid can activate the expression of Myh7 and Pgc-1α through a dual pathway, thereby significantly improving mitochondrial respiratory efficiency and muscle fiber contractile function, achieving the effects of enhancing athletic performance, promoting muscle growth, and alleviating exercise fatigue. This invention further validates through animal experiments that sialic acid can promote muscle growth, increase muscle mass and muscle mass, improve grip strength, and significantly prolong the time spent running and swimming to exhaustion.

[0009] The applications described in this invention relate to enhancing physical performance or relieving exercise fatigue. As those skilled in the art know, exercise fatigue is not a disease, and enhancing physical performance is not a treatment of disease. Therefore, the applications of this invention do not involve disease diagnosis or treatment.

[0010] Furthermore, the composition comprises sialic acid and ω-6 fatty acid.

[0011] Furthermore, the ω-6 fatty acid includes one or more of LA, GLA, DGLA, ARA, or DPA; Preferably, the ω-6 fatty acid is ARA.

[0012] Arachidonic acid (ARA) is an n-6 polyunsaturated fatty acid composed of 20 carbon atoms. It is widely distributed in the phospholipids of cell membranes in all tissues and directly participates in the regulation of various enzymes, ion channels, and bioactive substances produced by cells. It plays a regulatory role in cellular and even systemic physiological functions and pathological processes, particularly in the nervous, immune, and skeletal muscle systems. ARA supplements are used in training to improve muscle mass, strength, and endurance, but they can also temporarily increase the acute inflammatory response to exercise stress, potentially amplifying inflammatory signals and leading to increased and prolonged post-exercise muscle soreness, swelling, and stiffness. Furthermore, long-term high levels of ARA intake, especially in the context of insufficient omega-3 fatty acid intake, may increase the risk of thrombosis and negatively impact vascular endothelial function.

[0013] This invention reveals a synergistic effect when sialic acid and ARA are used simultaneously. Transcriptome analysis elucidated the synergistic mechanism of the composition. Differential gene clustering analysis showed that the expression levels of genes related to maintaining muscle cell homeostasis and muscle cell proliferation were significantly higher than in the single-ingredient treatment group. The core metabolic pathways involved in the differential gene enrichment, namely PI3K-Akt, cytoskeleton in muscle cells, and mTOR, were the most significantly enriched pathways. These three pathways jointly regulate muscle cell survival and proliferation. Muscle cell proliferation and muscle contraction are core pathways at the biological process level, providing a structural and functional basis for improving athletic performance. This composition can further significantly enhance the effect of improving physical performance or alleviating exercise fatigue, while reducing the dosage of ARA and avoiding negative effects such as excessive inflammatory response.

[0014] Furthermore, the mass ratio of sialic acid to ω-6 fatty acid is (1~20):1; Preferably, the mass ratio of sialic acid to ARA is (5~15):1.

[0015] Furthermore, the composition also includes ω-3 fatty acids, including EPA and / or DHA; Preferably, the mass ratio of sialic acid to ω-3 fatty acid is 20:1 to 1:20.

[0016] Furthermore, the application includes any one or more of the following: (1) Promotes muscle growth or increases muscle mass; (2) Improve grip strength or prolong the duration of exhaustion exercises; (3) Reduce serum lactate, ammonia, creatine kinase or blood urea nitrogen levels after exercise to improve exercise fatigue; (4) Increase the expression of Myh7 or Pgc-1α.

[0017] In a second aspect, the present invention provides a method for enhancing athletic performance or relieving exercise fatigue, comprising: using sialic acid; preferably, the dosage of sialic acid is 20~120 mg / kg / d, more preferably 80~120 mg / kg / d.

[0018] Thirdly, the present invention provides a composition comprising: sialic acid and ω-6 fatty acid; wherein the mass ratio of sialic acid to ω-6 fatty acid is (9~11):1.

[0019] Furthermore, the mass ratio of sialic acid to ω-6 fatty acid is (9.5~10.5):1, and the ω-6 fatty acid is ARA.

[0020] As a preferred embodiment, the composition provided by the present invention consists of sialic acid and ω-6 fatty acid, wherein the mass ratio of sialic acid to ω-6 fatty acid is (9.5~10.5):1, and the ω-6 fatty acid is ARA.

[0021] Fourthly, the present invention provides a product comprising the aforementioned composition, wherein the product is a food, health product, or drug.

[0022] The present invention has the following beneficial effects: This invention reveals that sialic acid, used alone and in combination with ω-3 or ω-6 fatty acids, can activate Myh7 and Pgc-1α expression through a dual pathway, significantly improving mitochondrial respiratory efficiency and muscle fiber contractile function. Therefore, it is particularly suitable for athletes undergoing high-intensity training and post-training recovery, and can be taken long-term as a functional food or dietary supplement to effectively delay fatigue and accelerate physical recovery. Combining modern nutritional theories and metabolic regulation mechanisms, this composition provides a scientific solution for improving athletic performance by optimizing cellular energy supply and muscle fiber functional status, and has broad market application prospects. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is an experimental flowchart provided in an embodiment of the present invention.

[0025] Figure 2 These are the changes in body weight of each group of mice and the initial and final body weight results provided in the embodiments of the present invention.

[0026] Figure 3 The results of the effects of supplemented EPA oil, SA, and ARA on the muscles of mice undergoing exercise training, provided in the embodiments of the present invention, are as follows: (A) muscle weight; (B) muscle coefficient.

[0027] Figure 4 The results of the supplementary EPA oil, SA, and ARA provided in the embodiments of the present invention on the forelimb grip strength of mice undergoing exercise training are as follows: (A) grip strength; (B) relative grip strength.

[0028] Figure 5 The results provided in this embodiment of the invention show the effects of supplemental EPA oil, SA, and ARA on running displacement and time to exhaustion in mice undergoing exercise training.

[0029] Figure 6 The results of this invention’s embodiments show the effects of supplemented EPA oil, SA, and ARA on the exhaustive swimming time of mice during exercise training.

[0030] Figure 7 The results show the effects of supplemented EPA oil, SA, and ARA on the gastrocnemius muscle tissue of exercise-trained mice, as provided in the embodiments of the present invention.

[0031] Figure 8 The results of this invention’s embodiments show the effects of supplementing with EPA oil, SA, and ARA on serum lactate (A), ammonia (B), creatine kinase (CK) (C), glucose (D), and blood urea nitrogen (BUN) (E) levels after acute exercise.

[0032] Figure 9 This is a culture diagram of L6 rat skeletal muscle cells provided in an embodiment of the present invention.

[0033] Figure 10 This is a graph showing the results of differential gene KEGG enrichment analysis provided in an embodiment of the present invention.

[0034] Figure 11 This is a graph showing the results of differential gene GO enrichment analysis provided in an embodiment of the present invention. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0036] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0037] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0038] Example 1 This embodiment examines the application of EPA, sialic acid (SA), and arachidonic acid (ARA) in improving physical performance and alleviating exercise fatigue, including the following procedures: 1. Experimental reagents.

[0039] EPA algal oil (EPA content ≥10 wt%), N-acetylneuraminic acid (purity ≥98%), and arachidonic acid oil (ARA content ≥50 wt%) are all commercially available.

[0040] 2. Laboratory animals.

[0041] SPF-grade four-week-old male ICR mice, weighing 23-26 g, are commercially available and meet ethical requirements.

[0042] 3. Experimental methods ( Figure 1 (For illustrative purposes only).

[0043] Before the experiment, the mice were acclimatized for one week. All animals were fed standard laboratory feed and distilled water, with free access to food, and were housed in an environment with room temperature (20-26℃) and humidity of 40%-70%, using a 12-hour light / dark cycle (lights were on from 06:00 to 18:00 every day).

[0044] After a one-week acclimatization period, the mice were randomly divided into 11 groups (n=8 per group): Resting control + vector group (NC).

[0045] Swimming training + vehicle group (M).

[0046] Swimming training + low-dose EPA algal oil group (LEPA).

[0047] Swimming training + medium-dose EPA algal oil group (MEPA).

[0048] Swimming training + high-dose EPA algal oil regimen (HEPA).

[0049] Swimming training + low-dose sialic acid (LSA) group.

[0050] Swimming training + medium-dose sialic acid (MSA) group.

[0051] Swimming training + high-dose sialic acid (HSA) group.

[0052] Swimming training + low-dose arachidonic acid (LARA) group.

[0053] Swimming training + medium-dose arachidonic acid group (MARA).

[0054] Swimming training + high-dose arachidonic acid (HARA) group.

[0055] During the experiment, mice in the NC and M groups were administered an equal volume of physiological saline via gavage daily. Mice in the LEPA, MEPA, HEPA, LSA, MSA, HSA, LARA, MARA, and HARA groups were administered corresponding doses of drugs (calculated as pure EPA, ARA, and SA substances) via gavage daily: 50 mg / kg / d EPA algal oil, 100 mg / kg / d EPA algal oil, 200 mg / kg / d EPA algal oil, 25 mg / kg / d SA, 50 mg / kg / d SA, 100 mg / kg / d SA, 50 mg / kg / d arachidonic acid oil, 100 mg / kg / d arachidonic acid oil, and 200 mg / kg / d arachidonic acid oil, for 38 days. In addition, mice in the M, LEPA, MEPA, HEPA, LSA, MSA, HSA, LARA, MARA, and HARA groups underwent 20 minutes of high-intensity aerobic swimming training daily during the 28-day treatment period. Swimming training was conducted after gavage administration, with intervals of 30 minutes. Swimming exercises were performed in a circular plastic pool (8 cm radius, 30 cm depth, water temperature 25 ± 2℃). The entire experimental period lasted 38 days, during which mouse weight was recorded weekly. After 28 days, all mice underwent various experiments sequentially (forelimb grip strength test on day 30, running training on days 31-33, running exhaustion test on day 34, and swimming exhaustion test on day 36). Finally, on day 38, after swimming without weight for 15 minutes, the experimental animals were immediately sacrificed, muscle mass was measured, and relevant fatigue biochemical indicators were analyzed.

[0056] 4. Detection methods.

[0057] (1) Organ index analysis and muscle tissue collection: Mice were euthanized by cervical dislocation, and the liver was removed, rinsed with saline, blotted dry with filter paper, and weighed. The liver index is expressed as the ratio of liver mass to mouse body weight. At the same time, gastrocnemius and soleus muscle tissues were harvested, rinsed with saline, blotted dry with filter paper, and weighed for subsequent experiments. The muscle coefficient is expressed as the ratio of total muscle weight (including soleus and gastrocnemius muscles) to body weight.

[0058] (2) Mouse forelimb grip strength test: The Penncentury SNSEK mouse grip strength tester (model: Snsek-SZ-SCG30) was used to analyze the changes in grip strength among the groups. The forelimb grip strength test was conducted 30 minutes after drug administration on day 30. The principle of the test is: when the mouse's tail is pulled, the mouse will instinctively grasp the metal grid in front of it to prevent its body from being passively pulled backward. When the pulling force applied by the operator exceeds the mouse's maximum grip strength, the mouse will release the grid, and the instrument will automatically record the maximum grip strength reached before releasing the grid. During the test, the mouse was first placed on a platform, and a metal grid with a force sensor was placed in front of it. The height of the grip grid was adjusted so that it was at the same level as the mouse's forelimb. The operator held the base of the mouse's tail at 1 / 3 of the position and guided it to grasp the measuring device. Then, the operator slowly pulled horizontally in the opposite direction to the mouse, keeping the mouse's body horizontal with the sensor. When the mouse released the grid, the instrument recorded its grip strength value. The test was repeated three times for each mouse. The maximum pulling force (in grams) recorded at the end is used as the grip strength index of the mouse.

[0059] (3) Running performance test: Before exercise, the mice underwent a 3-day treadmill adaptation training: (Day 1: 10 minutes of running at 10 m / min speed and 0 incline; Day 2: 5 minutes of running at 10 m / min speed and 0 incline, followed by 5 minutes of running at 10 m / min speed and 5° incline; Day 3: 5 minutes of running at 10 m / min speed, followed by 5 minutes of running at 15 m / min speed and 10° incline). To assess exercise ability, the mice were placed on a treadmill and warmed up for 5 minutes at 5° incline and 10 m / min speed, followed by 5 minutes of running at 10° incline and 10 m / min speed. The initial speed was set at 10 m / min and the incline at 10°. After running for 5 minutes, the speed was increased by 2 m / min every 5 minutes, reaching a maximum of 34 m / min. When the mouse received more than 7 electric shocks within 15 seconds, it was considered to be at the fatigue endpoint. The exercise time and the total distance run to reach fatigue were recorded.

[0060] (4) Swimming performance test: A swimming exhaustion test was conducted 30 minutes after drug administration on day 36. Mice were placed individually in a columnar swimming pool (container height 65 cm, radius 20 cm), with a water depth maintained at 40 cm and a water temperature controlled at 24 ± 1℃. A weight equivalent to 5% of the mouse's body weight was attached to the base of each mouse's tail, and the duration from the start of swimming to exhaustion was recorded. Exhaustion was defined as the mouse's inability to continue swimming, manifested by ceasing paddling, floating on the surface, struggling, until complete loss of strength and signs of sinking. Exhaustion was defined as when a mouse could not surface for air within 7 seconds. The time required for a mouse to swim to exhaustion was recorded as an indicator of its athletic performance.

[0061] (5) Histological analysis: After the mice were sacrificed, the gastrocnemius muscle was collected and fixed in 4% paraformaldehyde solution. After fixation, the tissue was embedded in paraffin and cut into sections with a thickness of 4 μm for morphological evaluation. The tissue sections were stained with hematoxylin and eosin (H&E), and the tissue morphology was observed under an optical microscope (Olympus, Tokyo, Japan).

[0062] (6) Determination of fatigue-related biochemical indicators: The effects of EPA algal oil supplementation, sialic acid, or arachidonic acid on fatigue-related biochemical indicators after exercise were assessed to accurately reflect and evaluate physiological status. Fatigue-related indicators were measured under fasting conditions to reflect the actual physiological status under exercise intervention. The effects of EPA algal oil, sialic acid, or arachidonic acid on serum levels of lactate, ammonia, glucose, blood urea nitrogen (BUN), and creatine kinase (CK) after exercise were evaluated. All mice were fasted for 12 h before the 15-minute swimming test. Before the test, mice were supplemented with EPA algal oil, sialic acid, or arachidonic acid, and the 15-minute weightless swimming test began 1 h later. Blood samples were collected immediately after the swimming exercise, centrifuged at 1500×g for 15 min at 4°C, and serum was collected for analysis. Fatigue-related biochemical indicators in serum were determined according to the kit instructions provided by Nanjing Jiancheng Biotechnology Institute (Nanjing, China).

[0063] 5. Results and Analysis.

[0064] (1) Effects of supplementing with EPA algal oil, sialic acid or arachidonic acid on the body weight of exercise-trained mice.

[0065] Mouse body weight was recorded weekly during the experiment to assess the effects of EPA algal oil, sialic acid, or arachidonic acid on mouse body weight. Results are shown below. Figure 2 After 4 weeks of aerobic exercise training or supplementation with EPA oil, SA, and ARA, the final body weights of mice in each group at the end of the experiment were 41.9±4.4 g, 39.2±3.0 g, 42.6±2.9 g, 41.5±3.3 g, 41.6±4.2 g, 43.2±2.8 g, 41.3±2.0 g, 41.3±3.2 g, 41.5±3.0 g, 41.8±3.9 g, and 40.2±1.6 g, respectively, with no statistically significant differences between groups. p >0.05).

[0066] (2) Effects of EPA algal oil supplementation, sialic acid or arachidonic acid on muscle mass and muscle coefficient in exercise-trained mice.

[0067] The effects of EPA algal oil, sialic acid, or arachidonic acid on muscle mass and its relative tissue weight (different tissue weights were adjusted for as a percentage of individual body weight) were evaluated. Results are shown in [Table missing]. Figure 3 Compared with the NC group, the muscle (gastrocnemius and soleus) mass of the LSA, MSA, HSA, MARA, and HARA groups was significantly increased. p <0.05), which increased muscle mass by 1.47 times, 1.36 times, 1.43 times, 1.47 times, and 1.38 times respectively; however, there was no statistically significant difference in muscle mass among the exercise groups. p >0.05); compared with the NC group, the muscle coefficients of the exercise control group and the drug treatment group increased by 1.39 times ( p <0.05), 1.11 times, 1.21 times, 1.28 times, 1.45 times ( p <0.05), 1.38 times ( p <0.05), 1.48 times ( p <0.05), 1.35 times ( p <0.05), 1.49 times ( p <0.05) and 1.45 times ( p <0.05); however, there was no significant difference in muscle coefficient between the exercise control group and the drug administration group ( p >0.05), indicating that supplementation with EPA, SA, and ARA under regular exercise intervention can promote muscle growth in mice.

[0068] (3) The effect of supplementing with EPA algal oil, sialic acid or arachidonic acid on the forelimb grip strength of mice undergoing exercise training.

[0069] Grip strength testing is a method used to assess neuromuscular coordination, muscle strength, and changes in overall functional ability. The forelimb grip strength and relative forelimb grip strength of mice in each group were measured and calculated. The results are shown below. Figure 4 The forelimb grip strength of the mice in each group was found to be 97.5±18.3 g, 113.8±25.6 g, 102.5±19.1 g, 126.3±20.7 g, 142.5±29.2 g, 110±17.7 g, 131.3±27.5 g, 170±22 g, 112.5±12.8 g, 141.3±24.2 g, and 165±21.4 g, respectively. Compared with the NC group, the forelimb grip strength of the M group was increased by 1.17 times. With the increase of drug dosage, the absolute forelimb grip strength increased in a dose-dependent manner. Among them, the forelimb grip strength of the HEPA group, HSA group, MARA group, and HARA group was significantly increased compared with the NC group. p <0.05), which increased by 1.46 times, 1.74 times, 1.45 times and 1.69 times respectively, and the forelimb grip strength in the HSA and HARA groups was significantly increased compared with the M group ( p<0.05). Grip strength data were corrected for individual body weight, and relative grip strength (%) was calculated. The results showed that the relative forelimb grip strength after HSA (411.4±48.1%) and HARA (411.1±51.9%) treatments was still significantly higher than that of the NC group (234.2±48.1%) and the M group (291.2±69.1%). p <0.05), indicating that under conditions of regular training intervention, EPA, SA, and ARA help to improve grip strength.

[0070] (4) The effects of supplementing with EPA algal oil, sialic acid or arachidonic acid on running displacement and running exhaustion time in mice undergoing exercise training.

[0071] Exhaustion running time and running distance are important parameters for assessing muscle strength and motor ability in mice. Compared with the NC group, the running displacement of each group increased by 2.16 times, 2.58 times, 3.33 times, 4.18 times, 2.35 times, 3.53 times, 4.21 times, 3.06 times, 3.57 times, and 4.35 times, respectively; among them, the running displacement of the MEPA, HEPA, MSA, HSA, LARA, MARA, and HARA groups was significantly increased ( p <0.05). The running exhaustion times of mice in the NC group, M group, and each drug-treated group were 15.8 min, 28 min, 32.2 min, 37.3 min, 43.8 min, 29.9 min, 39.4 min, 44.7 min, 36 min, 40.1 min, and 45.7 min, respectively; compared with the NC group, low, medium, and high doses of EPA, SA, and ARA administration significantly prolonged the running exhaustion time ( p <0.05), but compared with group M, only the high-dose drug significantly improved running endurance ( Figure 5 The results showed that EPA, SA, and ARA significantly enhanced muscle strength and improved motor performance in mice, especially in the high-dose group. Both exercise and drug administration had significant main effects on muscle endurance, and there was also a significant interaction between the two.

[0072] (5) The effect of supplementing with EPA algal oil, sialic acid or arachidonic acid on the exhaustion swimming time of mice during exercise training.

[0073] Exercise endurance is an important indicator for evaluating the effectiveness of sports nutrition. Swimming test results show that ( Figure 6Compared with the NC group, the swimming exhaustion time in the exercise control group and each drug-treated group increased, with the exhaustion swimming time increasing by 2.12 times, 1.99 times, 2.75 times, 4.60 times, 2.24 times, 2.96 times, 5.15 times, 2.65 times, 4.16 times, and 5.59 times, respectively. Among these, under high-dose supplementation with EPA, SA, and ARA, the mice's athletic performance was significantly prolonged compared to the NC and M groups. p <0.05). The results showed that under exercise training intervention, EPA, SA, and ARA improved exercise performance in a dose-dependent manner, and EPA, SA, and ARA supplementation had a synergistic effect on promoting exercise with aerobic exercise training.

[0074] (6) Effects of EPA algal oil supplementation, sialic acid, or arachidonic acid on the structure and morphology of muscle tissue in exercise-trained mice: HE staining analysis was performed on the gastrocnemius muscle of mice, and the results are shown in […]. Figure 7 From an overall morphological perspective, the gastrocnemius muscle fibers in each group were intact, clearly structured, and had regular outlines, with no obvious necrosis or breakage, indicating that the treatments did not produce adverse pathological changes in the muscle tissue. Compared with the NC and M groups, the muscle fibers in the EPA, SA, and ARA supplementation groups showed varying degrees of thickening trend.

[0075] (7) Effects of supplementing with EPA algal oil, sialic acid or arachidonic acid on fatigue-related biochemical indicators in exercise-trained mice.

[0076] The results are as follows Figure 8 As shown, exercise-induced muscle fatigue can be assessed using biochemical indicators such as lactate, ammonia, glucose, creatine kinase (CK), and blood urea nitrogen (BUN). Lactic acid accumulates in the blood and muscles involved in exercise, and buildup occurs when its production exceeds the aerobic metabolic capacity. When lactate concentration increases, hydrogen ions accumulate, leading to tissue acidification and thus fatigue. The lactate levels in each group were 13.47 ± 4.01, 12.51 ± 1.57, 10.20 ± 1.01, 9.32 ± 1.30, 7.92 ± 1.97, 10.30 ± 1.78, 8.90 ± 1.03, 8.38 ± 1.12, 9.79 ± 1.48, 9.07 ± 1.11, and 6.37 ± 1.15 mmol / L, respectively. Compared with the control group, the lactate levels in the LEPA group, MEPA group, HEPA group, LSA group, MSA group, HSA group, LARA group, MARA group, and HARA group were significantly reduced. p <0.05), respectively decreasing by 24.23%, 30.79%, 41.20%, 23.51%, 33.92%, 37.78%, 27.32%, 32.62%, and 52.67% ( Figure 8(A) Compared with group M, medium and high doses of EPA, SA, or ARA supplementation significantly reduced serum lactate levels (A). p <0.05). Trend analysis showed that serum lactate levels decreased in a dose-dependent manner with increasing EPA, SA, and ARA dosages. After acute exercise, the rate of blood lactate clearance has a significant impact on the recovery process; approximately 75% of lactate can be utilized for energy through oxidation or for glucose resynthesis in the liver. These results suggest that EPA, SA, or ARA supplementation may help with the clearance and utilization of blood lactate after exercise.

[0077] Ammonia is another important metabolic byproduct of energy metabolism during exercise, and its sources are diverse. The accumulation of ammonia in the blood and brain during exercise can negatively impact the central nervous system and cause fatigue. Although exercise-induced ammonia toxicity is usually temporary and reversible, it can still affect the sustained coordinated activity of key areas of the central nervous system. The central nervous system plays a crucial role in the development of physical fatigue. Supplementation with EPA, SA, or ARA may affect fatigue states by modulating the central nervous system. The serum ammonia levels in each group were 383.37 ± 36.52, 378.18 ± 72.95, 248.50 ± 31.44, 225.93 ± 44.42, 215.25 ± 27.28, 267.11 ± 41.07, 257.71 ± 15.53, 257.34 ± 40.40, 327.71 ± 67.33, 313.99 ± 62.14, and 261.58 ± 69.07 µmol / L, respectively. Figure 8 (B in the text). Compared with the control group, the LEPA group, MEPA group, HEPA group, LSA group, MSA group, HSA group, LARA group, MARA group, and HARA group showed reductions of 35.18%, 41.07%, 43.85%, 30.33%, 32.78%, 32.87%, 14.52%, 18.10%, and 31.77%, respectively. Compared with the NC group and M group, low, medium, and high doses of EPA and SA, as well as high doses of ARA supplementation, all significantly reduced serum ammonia levels (B in the text). p <0.05). Trend analysis showed that serum ammonia levels decreased significantly in a dose-dependent manner with increasing EPA, SA, and ARA doses, indicating that continuous supplementation with EPA, SA, or ARA can reduce ammonia accumulation during exercise.

[0078] Serum CK levels are important clinical biomarkers for muscle injury, muscular dystrophy, severe muscle breakdown, myocardial infarction, autoimmune myositis, and acute renal failure. High-intensity exercise can cause tissue damage and muscle cell necrosis through physical or chemical means. Normally, serum CK levels are low, but they increase when exercise-induced hypoxia and the accumulation of metabolic products lead to muscle cell damage, thereby reducing athletic performance. In this study, the serum CK levels in each group were 33.27 ± 4.72, 32 ± 7.78, 19.58 ± 3.32, 20.77 ± 4.2, 17.84 ± 4.19, 25.78 ± 4.9, 20.09 ± 4.31, 19.23 ± 3.6, 32.67 ± 6.23, 28.37 ± 3.3, and 27.49 ± 5.6 ng / mL, respectively. Figure 8 (C in the text). Compared with the control group, the LEPA group, MEPA group, HEPA group, LSA group, MSA group, HSA group, LARA group, MARA group, and HARA group showed reductions of 41.14%, 37.57%, 46.37%, 22.50%, 39.62%, 42.19%, 1.78%, 14.73%, and 17.35%, respectively. Compared with the NC group and M group, low, medium, and high doses of EPA and SA supplementation significantly reduced serum CK levels (C in the text). p <0.05), indicating that EPA and SA supplementation can improve acute exercise-induced skeletal muscle injury.

[0079] Blood glucose levels are an important indicator for maintaining athletic performance. The serum glucose levels in each group were 3.81 ± 0.95, 3.18 ± 1.44, 5.48 ± 1.33, 3.5 ± 1.31, 3.77 ± 1.32, 3.42 ± 1.13, 3.23 ± 0.71, 3.53 ± 1, 2.99 ± 0.96, 2.52 ± 0.71, and 2.86 ± 0.59 mmol / L, respectively. There were no statistically significant differences among the groups. Figure 8 (D in the middle).

[0080] Blood urea nitrogen (BUN) is an important biochemical indicator associated with fatigue, reflecting the nitrogen content in the blood derived from urea. Urea plays a crucial role in the metabolism of nitrogenous compounds. Elevated BUN levels suggest increased protein breakdown, which may weaken muscle contractility and lead to fatigue. In this study, the serum BUN levels in each group were 72.88 ± 13.2, 56.28 ± 8.23, 52.84 ± 8.31, 46.26 ± 3.36, 34.61 ± 5.33, 44.11 ± 9.51, 45.78 ± 10.72, 38.9 ± 6.13, 40.67 ± 10.35, 36.16 ± 6.37, and 35.05 ± 3.39 ng / mL, respectively. Figure 8 Compared with the control group, the BUN levels in the M group, LEPA group, MEPA group, HEPA group, LSA group, MSA group, HSA group, LARA group, MARA group, and HARA group were significantly reduced by 22.78%, 27.5%, 36.53%, 52.52%, 39.48%, 37.19%, 46.63%, 44.2%, 50.39%, and 51.91%, respectively. Compared with the M group, supplementation with ARA and high doses of EPA and SA significantly reduced serum BUN levels (E). p <0.05 indicates that supplementation with EPA, SA, or ARA can reduce BUN levels after acute exercise.

[0081] Example 2 1. Culture of L6 rat skeletal muscle cells.

[0082] (1) Remove or discard the original culture medium in the culture bottle.

[0083] (2) Wash the cells with PBS buffer 1-2 times.

[0084] (3) Add 1 mL of 0.25% EDTA-containing trypsin solution to the bottle.

[0085] (4) Place the culture flask in a 37°C incubator or at room temperature (25°C) for digestion. After 1-2 min, place the culture flask under a microscope for observation. When the cytoplasm shrinks and the intercellular spaces increase, add 1 mL of complete culture medium to stop the digestion.

[0086] (5) Use a pipette to gently blow the cells off the bottle wall repeatedly to form a cell suspension. Centrifuge at 1200 rpm for 5 min. After centrifugation, remove the supernatant and resuspend in 1 mL of complete culture medium.

[0087] (6) After counting with a counting chamber, the cells were seeded into new well plates and cultured in a CO2 incubator. After 24 h, the original culture medium was discarded, and complete culture medium containing 2% horse serum was added for differentiation culture. The culture diagram of L6 rat skeletal muscle cells is shown below. Figure 9 As shown.

[0088] 2. RNA extraction.

[0089] Sample: SA is a commercially available sialic acid product from Zhongke Optics Valley with a purity of 99%; ARA is a commercially available ARA oil from the applicant. Since the oil also contains other fatty acid components, it was calculated as pure ARA in the experiment.

[0090] Total RNA extraction from cells (Trizol method): Total RNA was extracted from cells using the Trizol lysis method. Glass tissue grinding rods, scissors, and other equipment used in the RNA extraction experiment were wrapped in aluminum foil and baked at 180°C for at least 4 hours. EP tubes and pipette tips were all RNase-free. The specific steps are as follows: (1) Sample preparation: L6 cell samples were prepared by adding 0.5% SA, 0.05% ARA, 0.2% ARA, and 0.25% SA + 0.025% ARA to a pH of 7.0. After washing the cells twice with basal medium, 1 mL of Trizol was added to each well of a 6-well plate to lyse the cells. The plates were then placed on a shaker and shaken at room temperature for 5-10 min.

[0091] (2) After the cells were fully lysed, they were transferred to 1.5 mL of RNase-free EP tubes. 200 µL of chloroform was added at a ratio of 200 µL chloroform / 1 mL Trizol. The tubes were vortexed vigorously for 15 s, placed on ice for 5 min, and centrifuged at 12000 rpm at low temperature (4℃) for 15 min.

[0092] (3) After centrifugation, the sample will show three layers: the bottom layer is the organic phase, the middle layer is the protein phase, and the top layer is the colorless aqueous phase (mainly containing RNA). Carefully aspirate the top aqueous phase into a new RNase-free EP tube (do not aspirate the middle white layer).

[0093] (4) Add an equal volume of isopropanol, gently shake to mix 6-8 times, place on ice for 10 min, and centrifuge at 12000 rpm and low temperature (4℃) for 15 min.

[0094] (5) Discard the supernatant; the white, transparent precipitate is RNA. Add 1 mL of pre-cooled 75% ethanol washing buffer (prepared with DEPC water) to each tube, gently invert to wash the precipitate thoroughly, and centrifuge at 8000 rpm at low temperature (4℃) for 5 min. Repeat twice.

[0095] (6) After discarding the supernatant, place it in a fume hood to dry for about 30 minutes.

[0096] (7) Add an appropriate amount of DEPC water according to the amount of RNA precipitation, and incubate it in a 55°C water bath for 7 minutes until it is fully dissolved in DEPC water.

[0097] (8) Determine the RNA concentration and purity, requiring the OD260 / OD280 ratio to be between 1.8 and 2.0. Take about 500 ng of RNA for reverse transcription experiments, and store the remaining RNA at -80℃ for subsequent experiments.

[0098] (9) Reverse transcription.

[0099] RNA reverse transcription was performed according to the Vazyme kit instructions, as follows: i) Residual genomic DNA removal.

[0100] Table 1 Residual Genomic DNA Removal System

[0101] Gently mix with a pipette and incubate at 42°C for 2 minutes using a PCR instrument.

[0102] ii) RNA reverse transcription.

[0103] Table 2 RNA reverse transcription system

[0104] Gently pipette to mix, then incubate at 50°C for 15 min and 85°C for 5 s using a PCR instrument.

[0105] The reaction product can be used immediately for qPCR, or stored at -20°C and used within six months; for long-term storage, it is recommended to aliquot and store at -80°C. Repeated freeze-thaw cycles should be avoided with cDNA.

[0106] Total RNA was isolated from transfected cells using Trizol reagent (Invitrogen) according to the manufacturer's instructions, as described above. Reverse transcription polymerase chain reaction (RT-PCR) was performed using a cDNA reverse transcription kit (Takara, Japan). Primers for detecting spliced ​​and unspliced ​​mRNA were designed according to previous research. The levels of spliced ​​and unspliced ​​mRNA were detected using the SYBR Green Master Mix Kit (Vazyme, China), with 2... -△△Ct The fold change was calculated using GAPDH (rat) as an endogenous control. The results are shown in Tables 5 and 6.

[0107] In this experiment, the levels of Myh7 and Pgc-1α genes were used as evaluation indicators, as they directly reflect the levels of their corresponding myosin and β-MHC proteins. Generally, due to complex regulation, gene levels cannot directly represent phenotypic levels, but Myh7 and Pgc-1α are exceptions. The former is a structural gene, and the latter is a master regulatory switch; changes in their expression levels often directly reflect changes in phenotype. Myosin heavy chain gene 7 (Myh7) directly encodes myosin "myosin heavy chain 7" (β-MHC protein), a core component of myosin. Therefore, when gene expression increases, the content of its direct translation product—β-MHC protein—naturally increases linearly. The most direct phenotype is an increase in the proportion of slow-twitch muscle fibers, resulting in enhanced muscle endurance. Conversely, downregulation means a decrease in slow-twitch muscle fibers. Peroxisome proliferator-activated receptor gamma coactivator 1α (Pgc-1α) is a key upstream regulatory gene for myosin expression. It is a core transcriptional coactivator controlling the characteristics of slow-twitch muscle fibers, driving the conversion of skeletal muscle towards slow, oxidative (type I) muscle fibers. Its expression is higher in slow-twitch fibers, and when its level increases, it synergistically interacts with proteins such as Mef2 to initiate a series of gene expression programs characteristic of slow-twitch fibers, including the upregulation of Myh7. In existing technologies, Myh7 and Pgc-1α are often directly used as biomarkers to evaluate the effects of various interventions on muscle performance and metabolism. For example, studies such as "Decreased Peak Expiratory Flow Associated with Muscle Fiber-Type Switching in Spinal and Bulbar Muscular Atrophy" and "Tbx1 regulates inherited metabolic and myogenic abilities of progenitor cells derived from slow- and fast-type muscle" both use them as indicators of muscle strength.

[0108] i) Reaction system.

[0109] Table 3 Reaction System

[0110] ii) Reaction conditions.

[0111] Table 4 Reaction conditions

[0112] Table 5. Effects of SA and ARA combination on Pgc-1α gene expression in L6 cells.

[0113] The significance level was 0.25%SA + 0.025%ARA compared to other groups.

[0114] Table 6. Effects of SA and ARA combination on Myh7 gene expression in L6 cells.

[0115] As shown in Tables 5 and 6, when SA and ARA were added alone, the expression levels of Pgc-1α and Myh7 genes in L6 cells were significantly higher than those in the control group. When the compound of 0.25% SA + 0.025% ARA was added, the expression levels of both genes were significantly higher than those in the control group, and the increase was greater than that of the two genes used alone. This indicates that when a certain proportion of the compound is added, SA and ARA work synergistically to further enhance mitochondrial function in muscles and relieve exercise fatigue.

[0116] Example 3 This invention performs transcriptome analysis on the above-mentioned 0.5% SA, 0.05% ARA, and 0.25% SA + 0.025% ARA sample groups. RNA concentration was accurately measured using a Qubit 4.0 fluorometer / MD microplate reader, and RNA integrity was detected using a Qsep 400 bioanalyzer. Oligo(dT) magnetic beads were used to enrich mRNA with poly A tails. After fragmentation, double-stranded cDNA was synthesized using random six-base primers. End repair, A-tailing, sequencing adapter ligation, fragment selection, and PCR enrichment were performed to obtain the final cDNA library. Preliminary quantification was performed using the Qubit dye method, and insert size was detected using a fragment analyzer. Libraries meeting the expected specifications were sequenced. Qualified libraries were merged and loaded onto the Illumina HiSeq X sequencing platform for high-throughput sequencing based on sequencing-by-synthesis (SBS) technology.

[0117] Using Hisat 2 version 2.2.1 with default parameters, sequencing reads were aligned with the rat reference genome mRatBN7.2.109, and the aligned reads were normalized to per million reads. Gene expression was quantified using feature counts version 2.0.3. Differential gene expression analysis was performed using R version 3.40.2 and DESeq 2 version 1.38.3. rMATS was used to identify and screen for significant differences based on the following criteria: |ΔPSI|>0.05 and false discovery rate (FDR)<0.05.

[0118] (1) Differential gene KEGG enrichment analysis.

[0119] KEGG pathway enrichment analysis was used to reveal the core metabolic and signaling pathways involved in differentially expressed genes. The results are presented in a bar chart. Figure 10 Presented.

[0120] KEGG enrichment bar charts and bubble charts can visually display the pathways to which differentially expressed genes are enriched in the combined sample. The horizontal axis represents the number of differentially expressed genes enriched in that pathway, and the vertical axis represents the name of the enriched pathway. The closer the bar color is to red, the higher the statistical significance; the size of the bubble chart points corresponds to the number of differentially expressed genes enriched, and the color intensity corresponds to the p-value (the darker the color, the higher the statistical significance).

[0121] The differential gene KEGG enrichment analysis results showed that the PI3K-Akt pathway had the most differentially enriched genes in the bar chart, significantly higher than other pathways. The bubble chart showed the largest and darkest dots, indicating that it was the pathway with the highest enrichment significance and correlation among all pathways. As the "core switch" regulating the biological functions of muscle cells, the activation of this pathway can directly promote the proliferation and differentiation of L6 cells and inhibit apoptosis. It can also regulate a variety of intracellular biological processes by phosphorylating a series of substrates, such as mTOR, one of the most common downstream effectors.

[0122] The high enrichment of differentially expressed genes in the cytoskeleton pathway in muscle cells indicates that the combination treatment can regulate actin polymerization, optimize the dynamic stability and flexibility of the cytoskeleton, reduce structural damage caused by mechanical stress during muscle movement, and improve the coordination of muscle fiber contraction.

[0123] The mTOR pathway, a key pathway for regulating protein synthesis and energy metabolism, can regulate the synthesis of ribosomes and the translation of proteins by sensing the energy status of cells. As shown in the figure, more than 12 differentially expressed genes are enriched in this pathway, and the enrichment significance reaches the core level, indicating that the combination treatment can significantly improve the energy metabolism efficiency of muscle cells.

[0124] (2) GO enrichment analysis of differentially expressed genes.

[0125] GO functional enrichment analysis analyzed differentially expressed genes from three dimensions: biological process (BP), cellular component (CC), and molecular function (MF). The results were presented in a bar chart. Figure 11 Presented.

[0126] GO enrichment bar charts and bubble charts can visually display the pathways to which differentially expressed genes are enriched in the combined sample group. The horizontal axis represents the number of differentially expressed genes enriched in that pathway, and the vertical axis represents the name of the enriched pathway. The closer the bar color is to red, the higher the statistical significance; the size of the bubble chart points corresponds to the number of differentially expressed genes enriched, and the color intensity corresponds to the P-value (the darker the color, the higher the statistical significance).

[0127] The differential gene GO enrichment analysis results showed that, at the biological process (BP) level, muscle cell proliferation and muscle contraction, two movement-related pathways, were among the top 5 pathways with the highest number of enriched differential genes, and both were prominent core pathways in the figure. This indicates that after the combination treatment, a large number of genes related to muscle cell proliferation and muscle contraction underwent significant expression changes. The increase in the number of muscle cells can enhance the overall volume and endurance of muscle tissue, which is the structural basis for improved exercise performance. Muscle contraction is the core physiological process of exercise execution. The significant enrichment of differential genes suggests that the combination treatment may further improve the efficiency and coordination of muscle movement by optimizing muscle cell contraction function.

[0128] The results above suggest that when sialic acid and ARA are used together, they may primarily enhance physical performance and alleviate exercise fatigue by regulating various metabolic pathways related to muscle cells.

[0129] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0130] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of sialic acid, or a composition containing sialic acid, in any of the following: (1) Enhance athletic ability or relieve exercise fatigue; (2) Prepare products for enhancing athletic ability or relieving exercise fatigue.

2. The application according to claim 1, characterized in that, The composition comprises sialic acid and ω-6 fatty acid.

3. The application according to claim 2, characterized in that, The ω-6 fatty acids include one or more of LA, GLA, DGLA, ARA, or DPA; Preferably, the ω-6 fatty acid is ARA.

4. The application according to claim 2 or 3, characterized in that, The mass ratio of sialic acid to ω-6 fatty acid is (1~20):1; Preferably, the mass ratio of sialic acid to ARA is (5~15):

1.

5. The application according to any one of claims 1-4, characterized in that, The composition further includes ω-3 fatty acids, including EPA and / or DHA; Preferably, the mass ratio of sialic acid to ω-3 fatty acid is 20:1 to 1:

20.

6. The application according to any one of claims 1-5, characterized in that, The applications include any one or more of the following: (1) Promotes muscle growth or increases muscle mass; (2) Improve grip strength or prolong the duration of exhaustion exercises; (3) Reduce serum lactate, ammonia, creatine kinase or blood urea nitrogen levels after exercise to improve exercise fatigue; (4) Increase the expression of Myh7 or Pgc-1α.

7. A method for enhancing athletic performance or relieving exercise fatigue, characterized in that, include: Sialic acid is used; preferably, the dosage of sialic acid is 20~120 mg / kg / d.

8. A composition, characterized in that, include: Sialic acid and ω-6 fatty acid; the mass ratio of sialic acid to ω-6 fatty acid is (9~11):

1.

9. The composition according to claim 8, characterized in that, The mass ratio of sialic acid to ω-6 fatty acid is (9.5~10.5):1, and the ω-6 fatty acid is ARA.

10. A product characterized in that, The product includes the composition of claim 8 or 9, and the product is a food, health product, or drug.

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