An amino acid supplement composition based on exercise session and gender stratification and application thereof

Abstract

The application provides an amino acid supplement composition based on exercise period and gender stratification and application thereof, and belongs to the technical field of sports nutrition. The amino acid supplement composition provided by the application comprises amino acid supplement composition 1 composed of leucine, isoleucine, arginine and valine, which is suitable for male morning fasting exercise; amino acid supplement composition 2 composed of leucine, isoleucine, arginine, valine and glycine, which is suitable for female morning fasting exercise; amino acid supplement composition 3 composed of leucine, isoleucine and ornithine, which is suitable for male evening postprandial exercise; and amino acid supplement composition 4 composed of leucine and isoleucine, which is suitable for female evening postprandial exercise. The application provides a function-oriented amino acid supplement composition which is designed differently according to exercise period and gender and is based on the correlation analysis of amino acids and physiological indexes, and can achieve the purpose of 'one supplement, multiple benefits'.

Description

Technical Field

[0001] This invention belongs to the field of sports nutrition technology, specifically relating to an amino acid supplement composition based on exercise time and gender stratification and its application. Background Technology

[0002] The core objective of sports nutrition supplementation is not only to replenish depleted nutrients, but also to precisely regulate the physiological stress response induced by exercise, promote recovery, and enhance long-term adaptability. Amino acids, as important metabolic substrates and signaling molecules, not only participate in energy supply during exercise but also exert a wide range of physiological regulatory effects by influencing pathways such as heart rate, energy expenditure, glucose and lipid metabolism, and purine metabolism. Serum amino acids, as key indicators of protein metabolism and nutritional status, are widely involved in various physiological processes such as energy supply, immune function regulation, and neurotransmitter synthesis, playing a crucial role in maintaining metabolic homeostasis. In recent years, the dynamic changes in serum free amino acid profiles have gradually become a key entry point for revealing the body's metabolic state and intrinsic regulatory mechanisms, especially in the field of sports science, where analyzing the dynamic changes in amino acid profiles can further clarify the mechanisms by which the body adapts to exercise load. Exercise plays an irreplaceable role in improving metabolic function and slowing the progression of chronic metabolic diseases. However, the molecular mechanisms behind the health benefits of exercise are not yet fully understood, particularly systematic exploration at the level of overall amino acid metabolism remains relatively limited. Existing sports amino acid supplements have the following technical shortcomings: they only focus on "replenishing consumption" and do not focus on "regulatory function"; they do not use the correlation between amino acids and indicators such as heart rate, energy consumption, glucose and lipid metabolism, and uric acid to guide formula design; they do not differentiate the design for the different physiological regulatory needs of men and women after exercise; and they do not distinguish the focus of functional regulation in different scenarios such as fasting in the morning and after dinner in the evening. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to provide a functional amino acid supplement composition that is differentiated according to the exercise time (fasting in the morning / postprandial in the evening) and gender, and based on the correlation analysis of amino acids and physiological indicators, so as to achieve "one supplement for multiple benefits", that is, to both compensate for exercise consumption and actively regulate heart rate, glucose and lipid metabolism and uric acid levels.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an amino acid supplement composition stratified by exercise time and gender, comprising an amino acid supplement composition 1 suitable for men after morning fasting exercise, an amino acid supplement composition 2 suitable for women after morning fasting exercise, an amino acid supplement composition 3 suitable for men after evening post-meal exercise, and / or an amino acid supplement composition 4 suitable for women after evening post-meal exercise; wherein the amino acid supplement composition 1 is composed of the following active amino acid ingredients: leucine, isoleucine, arginine, and valine; wherein the amino acid supplement composition 2 is composed of the following active amino acid ingredients: leucine, isoleucine, arginine, valine, and glycine; wherein the amino acid supplement composition 3 is composed of the following active amino acid ingredients: leucine, isoleucine, and ornithine; and wherein the amino acid supplement composition 4 is composed of the following active amino acid ingredients: leucine and isoleucine.

[0005] Preferably, in the amino acid supplement composition 1, based on the daily dosage, there are 1.5-3 g of hydrolyzed leucine, 0.75-1.5 g of hydrolyzed isoleucine, 0.5-2 g of hydrolyzed arginine, and 0.75-1.5 g of hydrolyzed valine.

[0006] Preferably, in the amino acid supplement composition 2, based on the daily dosage, there are 1.5-3 g of hydrolyzed leucine, 0.75-1.5 g of hydrolyzed isoleucine, 0.5-2 g of hydrolyzed arginine, 0.75-1.5 g of hydrolyzed valine, and 1-3 g of hydrolyzed glycine.

[0007] Preferably, the amino acid supplement composition 3 contains, based on the daily dosage, 1.5-3 g of hydrolyzed leucine, 0.75-1.5 g of hydrolyzed isoleucine, and 0.5-2 g of hydrolyzed ornithine.

[0008] Preferably, the amino acid supplement composition 4 contains 1.5-3 g of hydrolyzed leucine and 0.75-1.5 g of hydrolyzed isoleucine, based on a daily dosage.

[0009] Preferably, the compound source of the leucine includes L-leucine; the compound source of the isoleucine includes L-isoleucine; the compound source of the arginine includes L-arginine, L-arginine hydrochloride, or L-arginine-aspartic acid; the compound source of the valine includes L-valine; the compound source of the glycine includes glycine; and the compound source of the ornithine includes L-ornithine hydrochloride.

[0010] Preferably, the dosage form of the amino acid supplement composition includes solid (powder, granules, tablets, blocks), semi-solid (gel, paste) or liquid.

[0011] The present invention also provides the application of the above-mentioned amino acid supplement composition in the preparation of products for post-exercise recovery, heart rate regulation, blood lipid regulation, blood glucose stabilization, ammonia metabolism regulation and / or uric acid management.

[0012] This invention also provides a method for amino acid supplementation after exercise, comprising the following steps: Subjects were categorized into four groups based on their exercise time, whether they exercised on an empty stomach, and their gender: men exercising on an empty stomach in the morning, men exercising after a meal in the evening, women exercising on an empty stomach in the morning, and women exercising after a meal in the evening. Then, the aforementioned amino acid supplement compositions were matched according to subject type: men took amino acid supplement composition 1 after exercising on an empty stomach in the morning, women took amino acid supplement composition 2 after exercising on an empty stomach in the morning, men took amino acid supplement composition 3 after exercising after a meal in the evening, and women took amino acid supplement composition 4 after exercising after a meal in the evening.

[0013] Preferably, the corresponding amino acid supplement composition is taken within 30 minutes after exercise, and the single dose is 0.5 to 2 g based on the total weight of the active amino acid ingredients.

[0014] The beneficial effects of this invention are: The amino acid supplement composition provided by this invention is a function-oriented design-based amino acid nutritional supplement composition. This invention is the first to directly translate the correlation analysis results of amino acids with heart rate, energy consumption, blood lipids, blood glucose, and uric acid into the basis for formula design, achieving a leap from "replenishing consumption" to "regulating function." This invention provides precisely matched supplementation solutions for four typical exercise scenarios: male / female and morning fasting / evening post-meal. Furthermore, based on the principle of "do not supplement if levels are already elevated," it excludes amino acids that have significantly increased after exercise, avoiding metabolic burden. Attached Figure Description

[0015] Figure 1 Principal component analysis plots for all samples and quality control samples.

[0016] Figure 2 Principal component analysis plot (A), partial least squares discriminant analysis plot (B), and random permutation test plot (C) are shown for different groups.

[0017] Figure 3 The diagram shows the principal component analysis (A), partial least squares discriminant analysis (B), and random permutation test (C) diagrams between motion and non-motion.

[0018] Figure 4 Principal component analysis plots (A), partial least squares discriminant analysis plots (B), and random permutation test plots (C) are shown for fasting in the morning and after dinner.

[0019] Figure 5Principal component analysis plots (A), partial least squares discriminant analysis plots (B), and random permutation test plots (C) are for males and females.

[0020] Figure 6 The trend of serum amino acid changes after exercise.

[0021] Figure 7 These are amino acids that are only related to exercise. .

[0022] Figure 8 Amino acids are the result of a combination of diet and exercise. .

[0023] Figure 9 To restore the amino acid intake lost during exercise through diet, .

[0024] Figure 10 Amino acids that show no change during fasting exercise but increase during postprandial exercise. .

[0025] Figure 11 Amino acids involved in sex-dietary interactions, .

[0026] Figure 12 This study analyzed the correlation between serum amino acids and blood biochemical parameters, heart rate, and energy expenditure after fasting exercise in the morning. A represents results for males, and B represents results for females. .

[0027] Figure 13 This study analyzed the correlation between serum amino acids and blood biochemical parameters, heart rate, and energy expenditure after fasting exercise in the evening. A represents results for males, and B represents results for females. . Detailed Implementation

[0028] This invention provides an amino acid supplement composition stratified by exercise time and gender, comprising an amino acid supplement composition 1 suitable for men after morning fasting exercise, an amino acid supplement composition 2 suitable for women after morning fasting exercise, an amino acid supplement composition 3 suitable for men after evening post-meal exercise, and / or an amino acid supplement composition 4 suitable for women after evening post-meal exercise; wherein the amino acid supplement composition 1 is composed of the following active amino acid ingredients: leucine, isoleucine, arginine, and valine; wherein the amino acid supplement composition 2 is composed of the following active amino acid ingredients: leucine, isoleucine, arginine, valine, and glycine; wherein the amino acid supplement composition 3 is composed of the following active amino acid ingredients: leucine, isoleucine, and ornithine; and wherein the amino acid supplement composition 4 is composed of the following active amino acid ingredients: leucine and isoleucine.

[0029] This invention systematically analyzed the changes of 24 serum amino acids in a population before and after exercise using UPLC-TQ MS technology. Correlation analysis revealed significant associations between these amino acid changes and indicators such as heart rate (HR), energy expenditure (EE), blood glucose (GLU), blood lipids (TC, HDL-C, LDL-C), and uric acid (UA). Key findings include: After exercising on an empty stomach in the morning: For men: Isoleucine was positively correlated with heart rate; valine was negatively correlated with triglycerides (TG); tyrosine and phenylalanine were negatively correlated with HDL-C; methionine was positively correlated with energy expenditure. For women: Tyrosine was negatively correlated with energy expenditure; methionine was negatively correlated with blood glucose; glycine was negatively correlated with LDL-C.

[0030] After exercise following an evening meal: In men: leucine and phenylalanine were positively correlated with heart rate; glycine, lysine, and aspartic acid were negatively correlated with total cholesterol (TC); ornithine was broadly positively correlated with TC, HDL-C, and LDL-C, and negatively correlated with energy expenditure. In women: leucine, isoleucine, tyrosine, and phenylalanine were negatively correlated with uric acid (UA); isoleucine was negatively correlated with blood glucose.

[0031] The aforementioned correlations reveal that amino acids are not only the targets for replenishment but also "functional molecules" that regulate physiological functions. However, existing sports amino acid supplements only focus on "what to supplement," neglecting "what to actively regulate through supplementation," and failing to incorporate gender- and exercise-time-specific physiological indicators into their formulation design. Based on the above correlation analysis, this invention proposes for the first time a "function-oriented" amino acid supplementation strategy: designing compositions containing targeted functional amino acids according to the specific physiological regulatory needs of different exercise times and genders, achieving "multiple benefits from one supplement"—compensating for exercise-related depletion while actively regulating heart rate, glucose and lipid metabolism, and uric acid levels.

[0032] In this invention, the design objective of the amino acid supplement composition 1 suitable for men after morning fasting exercise is to replenish energy expenditure and regulate heart rate and lipid metabolism. In the amino acid supplement composition 1, based on the daily dosage, the hydrolyzed leucine content is preferably 1.5~3 g, more preferably 1.5~2.5 g; the hydrolyzed isoleucine content is preferably 0.75~1.5 g, more preferably 1~1.5 g; the hydrolyzed arginine content is preferably 0.5~2 g, more preferably 1~2 g; and the hydrolyzed valine content is preferably 0.75~1.5 g, more preferably 1~1.5 g.

[0033] In this invention, the design objective of the amino acid supplement composition 2 suitable for women after morning fasting exercise is to replenish energy consumption and regulate energy consumption and glucose metabolism. In the amino acid supplement composition 2, based on the daily dosage, the hydrolyzed leucine content is preferably 1.5~3 g, more preferably 1.5~2.5 g; the hydrolyzed isoleucine content is preferably 0.75~1.5 g, more preferably 1~1.5 g; the hydrolyzed arginine content is preferably 0.5~2 g, more preferably 1~1.5 g; the hydrolyzed valine content is preferably 0.75~1.5 g, more preferably 1~1.5 g; and the hydrolyzed glycine content is preferably 1~3 g, more preferably 1.5~2.5 g.

[0034] In this invention, the design objective of the amino acid supplement composition 3 suitable for men after evening exercise is to replenish energy expenditure and regulate lipid and ammonia metabolism. The amino acid supplement composition 3, based on daily dosage, preferably contains 1.5-3 g of hydrolyzed leucine, more preferably 1.5-2.5 g; preferably 0.75-1.5 g of hydrolyzed isoleucine, more preferably 1-1.5 g; and preferably 0.5-2 g of hydrolyzed ornithine, more preferably 1-1.5 g. The amino acid supplement composition 3 provided by this invention contains ornithine, which can reduce blood ammonia levels after exercise and improve blood lipid profiles.

[0035] In this invention, the design objective of the amino acid supplement composition 4, suitable for women after evening exercise, is to replenish uric acid and regulate its metabolism. The amino acid supplement composition 4, based on a daily dosage, preferably contains 1.5-3 g of hydrolyzed leucine, more preferably 1.5-2.5 g, and preferably 0.75-1.5 g of hydrolyzed isoleucine, more preferably 1-1.5 g. The amino acid supplement composition 4 provided by this invention, composed of leucine and isoleucine, can help lower uric acid levels, and is particularly suitable for female athletes.

[0036] In this invention, the preferred source of the leucine compound is L-leucine; the preferred source of the isoleucine compound is L-isoleucine; the preferred source of the arginine compound is L-arginine, L-arginine hydrochloride, or L-arginine-aspartic acid; the preferred source of the valine compound is L-valine; the preferred source of the glycine compound is glycine; and the preferred source of the ornithine compound is L-ornithine hydrochloride. In this invention, the dosage form of the amino acid supplement composition includes solid (powder, granules, tablets, blocks), semi-solid (gel, paste), or liquid. This invention does not have any particular limitation on the excipients used to formulate the amino acid supplement composition into various dosage forms; conventional excipients in the art can be used.

[0037] The scientific basis for designing the above-mentioned amino acid supplement compositions in this invention is of a high level, and all design basis comes from statistically significant differences in randomized controlled trials. P <0.05~0.001) and correlation analysis (Pearson / Spearman correlation coefficient).

[0038] This invention also provides the application of the above-mentioned amino acid supplement composition in the preparation of products for post-exercise recovery, heart rate regulation, blood lipid regulation, blood glucose stabilization, ammonia metabolism regulation, and / or uric acid management. In this invention, the products preferably include pharmaceuticals, food products, or health supplements.

[0039] This invention also provides a method for amino acid supplementation after exercise, comprising the following steps: Subjects were categorized into four groups based on their exercise time, whether they exercised on an empty stomach, and their gender: men exercising on an empty stomach in the morning, men exercising after a meal in the evening, women exercising on an empty stomach in the morning, and women exercising after a meal in the evening. Then, the aforementioned amino acid supplement compositions were matched according to subject type: men took amino acid supplement composition 1 after exercising on an empty stomach in the morning, women took amino acid supplement composition 2 after exercising on an empty stomach in the morning, men took amino acid supplement composition 3 after exercising after a meal in the evening, and women took amino acid supplement composition 4 after exercising after a meal in the evening.

[0040] In the method described in this invention, it is preferred to take the corresponding amino acid supplement composition within 30 minutes after exercise. The dosage for a single dose is based on the total weight of the active amino acid ingredients, preferably 0.5-2g after exercise, more preferably 1-1.5g after exercise. The dosage described here is the dosage taken once after exercise. The total daily amino acid supplement should conform to the above-mentioned daily dosage by weight and the amount of each amino acid calculated by hydrolysis.

[0041] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0042] Unless otherwise specified, the following embodiments are all conventional methods.

[0043] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0044] Example 1 This invention targets healthy young students and employs a randomized controlled design, involving three days of moderate-intensity rope skipping exercise, one in the morning on an empty stomach and the other after a dinner meal. Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS / MS) was used to quantitatively determine the concentrations of 24 amino acids in serum before and after exercise. By comparing changes in serum amino acid profiles before and after exercise (exercise vs. non-exercise, morning on an empty stomach vs. evening after a dinner meal, and in men and women), and combining these findings with the correlations between amino acids and blood biochemical indicators, heart rate, and energy expenditure, the invention elucidates the influence of these factors on serum amino acid metabolism. This provides new evidence for a deeper understanding of the complexity of exercise adaptation mechanisms and offers a theoretical basis for personalized exercise and nutrition guidance that considers morning / evening exercise time, dietary status, and gender.

[0045] 1. Test Methods 1.1 Human Trial Design 1.1.1 Subject Recruitment This invention was reviewed and approved by the Ethics Review Committee of Harbin Medical University. A total of 35 healthy adult volunteers, aged 22-30 years, were recruited. The inclusion criteria for the subjects were as follows: (1) aged 18-30 years, with no history of major surgery or trauma; (2) basic physiological indicators (blood pressure, blood sugar, etc.) were all within the normal range; (3) no history of acute or chronic diseases or infectious diseases; (4) avoid using any dietary supplements or drugs that may affect metabolism during the study period. The exclusion criteria included: (1) a history of diagnosis of tumors or hereditary diseases; (2) use of antibiotics within the past month, or abnormal metabolic-related biochemical indicators. After screening, a total of 34 subjects were finally selected. All subjects signed informed consent forms before the trial and confirmed that there were no health risk factors that may affect physical performance (ChiCTR2200064349).

[0046] 1.1.2 Dietary Intake Assessment Participants ate at the cafeteria at 8:00, 12:00, and 18:00 daily, with consistent dietary choices for each meal. Apart from these three meals, participants did not consume any extra meals, snacks, milk tea, or beverages containing caffeine or alcohol. A 24-hour dietary questionnaire was used to continuously record the amount consumed at each meal, and a nutrition calculator v2.7.3 was used to assess energy and nutrient intake.

[0047] 1.1.3 Human Trial Protocol This study was conducted from November 28 to December 10, 2022, in the Sunshine Hall of the School of Public Health, Harbin Medical University. First, basic information of the participants (including age, gender, weight, etc.) was collected. Then, the participants were randomly divided into an exercise group (n=17) and a control group (n=17) using a 1:1 propensity score matching method. Each group included 6 males and 11 females.

[0048] Exercise monitoring: To explore the effect of moderate-intensity exercise on serum amino acid levels, all participants wore a Huawei Smart Band 7 during exercise to monitor their heart rate and record energy expenditure in real time, while ensuring that their heart rate remained within the target heart rate range (60-70% of their maximum heart rate). The target heart rate refers to the exercise heart rate range that can safely and effectively improve cardiovascular function, and is calculated as: Target heart rate = [(220 - age) - resting heart rate] × (60%–70%) + resting heart rate. When the heart rate exceeded the upper limit, the band would issue an alarm to prompt deceleration.

[0049] The exercise group underwent two days of acclimatization training before the formal experiment began, jumping rope at a speed of 60-70 jumps per minute for 40 minutes to gradually adapt to the exercise intensity. A seven-day washout period was set between the two formal exercise interventions.

[0050] Morning fasting exercise: The exercise group performed fasting rope skipping exercises for 40 minutes daily from 7:00 to 7:40 AM from November 28th to November 30th, for 3 consecutive days; the control group remained at rest during the same time period and did not engage in any exercise. For the rest of the time, the activity schedules of both groups were consistent, and both groups ate meals in the cafeteria.

[0051] Evening exercise after meals: The exercise group performed 40 minutes of skipping rope exercise daily from 19:00 to 19:40 one hour after meals from December 8th to December 10th, for 3 consecutive days; the control group remained at rest during the same time period and did not perform any exercise. During the rest of the time, the activity schedules of the two groups of subjects were the same, and both groups ate in the cafeteria.

[0052] 1.1.4 Sample Collection for Population Trials Heart rates of all subjects were recorded before and immediately after each exercise session, and energy expenditure of subjects was recorded immediately after exercise.

[0053] On the third day of continuous exercise intervention, venous blood was collected from all subjects by four staff members. Blood samples were collected from the median cubital vein of the subjects before exercise, immediately after exercise in the exercise group, and at the same time points in the control group. Immediately after collection, the blood samples were centrifuged at 3000 rpm for 10 minutes, and the supernatant serum was separated and stored in an ultra-low temperature freezer at -80°C for subsequent blood biochemical index detection and UPLC-TQ MS analysis.

[0054] 1.2 Serum biochemical marker detection The following 10 biochemical indicators in serum samples were measured using a fully automated biochemical analyzer: alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (UA), blood glucose (GLU), blood urea nitrogen (BUN), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and creatinine (CRE).

[0055] 1.3 Quantitative Detection and Analysis of Serum Amino Acids 1.3.1 Sample Pretreatment (1) Remove the serum sample from the -80℃ freezer, thaw it under gradient conditions, and vortex for 2 minutes. Take 30 μL of well-mixed serum into a 1.5 mL EP tube, add 80 μL of sample processing solution (methanol:acetonitrile:formic acid = 24.9:74.9:0.2), vortex for 30 seconds, and then store at -20℃ for 2 hours.

[0056] (2) Centrifuge at 15000 r / min for 15 minutes under low temperature conditions, aspirate the supernatant and transfer it to a new EP tube, and let it stand overnight at -20℃ (about 12 hours).

[0057] (3) The next day, centrifuge again under the same conditions for 15 min, take the supernatant and transfer it to a Waters sample bottle for LC-MS analysis.

[0058] Quality control (QC) sample preparation: Take 50 μL of supernatant from each sample and combine it into a new 5 mL centrifuge tube. Mix thoroughly to prepare a mixed QC sample. Perform the entire process on ice to minimize the impact of repeated freeze-thaw cycles on metabolite stability.

[0059] 1.3.2 UPLC-TQ MS Instrument Parameters Serum amino acids were quantitatively detected by UPLC-MS / MS using an ACQUITY ultra-high performance liquid chromatography system (Waters, USA) equipped with a Waters Xevo TQD mass spectrometer and selected reaction monitoring (SRM) in hydrophilic interaction chromatography (HILIC) mode. Instrument parameters are shown in Table 1. Mass spectrometric conditions and retention times for each amino acid are shown in Table 2.

[0060] Table 1 UPLC-TQ MS Instrument Parameters

[0061] Table 2 Mass spectrometry conditions and retention times for amino acids

[0062] 1.3.3 Quantitative analysis of amino acids The external standard method was used to quantify the amino acids in serum. First, standard solutions of different concentrations were prepared using amino acid standard substances. Then, by measuring the peak area of ​​the standards and establishing a linear relationship with known concentrations, a standard curve was established to calculate the absolute content of each amino acid in the serum sample. Within the linear range, the standard curves for all 24 amino acids showed good linearity (all R0). 2 The limits of detection (LOD) for each amino acid ranged from 0.002 to 0.058 μg / mL, and the limits of quantitation (LOQ) ranged from 0.004 to 0.118 μg / mL, all of which were lower than the lowest concentration point of the standard curve, meeting the requirements for quantitative analysis of serum samples.

[0063] Table 3 Standard curves and linear ranges of amino acids

[0064] Since the serum sample was diluted 4 times before testing, the actual plasma concentration is converted using the following formula: .

[0065] 1.4 Statistical Analysis Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed using MetaboAnalyst R package (Version 4.0) within R software (Version ≥ 4.2.0) to assess the clustering trends of amino acids within and between different groups. The established PLS-DA model was validated using a permutation test with 200 randomizations to evaluate its robustness and overfitting risk. GraphPad Prism 7.0 (GraphPad Software Inc. San Diego, CA, USA) was used for graphing and statistical analysis. Normally distributed measurement data, as determined by the Shapiro-Wilk test, are expressed as mean ± standard deviation (X ± S). Independent samples t-tests were used for comparisons between two groups; one-way ANOVA was used for comparisons among multiple groups. For values ​​that do not conform to a normal distribution, the Mann-Whitney U test or the Kruskal-Wallis test is used. Correlation analysis between variables is performed: Pearson correlation analysis is used for variables conforming to a normal distribution, and Spearman rank correlation analysis is used for variables not conforming to a normal distribution. All statistical tests are two-tailed tests. P <0.05 indicates a statistically significant difference. ).

[0066] 2. Results 2.1 Basic characteristics of the research subjects A total of 34 people participated in this study, including 12 men and 22 women. As shown in Table 4, there were no statistically significant differences in the basic characteristics between the exercise group and the control group. P >0.05).

[0067] Table 4 Basic physical indicators of the subjects

[0068] 2.2 Dietary Intake Assessment As shown in Table 5, there were no statistically significant differences between the exercise group and the control group in terms of energy, the three major nutrients (carbohydrates, fats, and proteins), vitamins, minerals, and dietary fiber intake. P >0.05).

[0069] Table 5 Comparison of dietary nutrient intake between the exercise group and the control group

[0070] 2.3 Serum biochemical parameters, heart rate, and energy expenditure before and after exercise As shown in Table 6, after exercising on an empty stomach in the morning, blood glucose levels decreased in both the exercise group and the control group, but the decrease was more significant in the exercise group. Furthermore, serum uric acid levels, heart rate, and energy expenditure all showed an upward trend in the exercise group. Notably, only the female exercise group showed a significant decrease in total cholesterol levels after exercise. P <0.05), no significant changes were observed in males ( P >0.05). As shown in Table 7, after evening post-dinner exercise intervention, both male and female exercise groups showed an increase in heart rate and energy expenditure, while the remaining serum biochemical indicators did not show significant differences between the exercise group and the control group. P >0.05).

[0071] Table 6. Blood biochemical indicators, heart rate, and energy expenditure during morning fasting exercise.

[0072] Table 7. Post-dinner exercise blood biochemical indicators, heart rate, and energy expenditure.

[0073] 2.4 Instrument stability assessment like Figure 1 As shown, the QC samples (red) are tightly clustered on the PCA score map, indicating that the instrument has good stability during the experiment.

[0074] 2.5 Analysis of differences in serum amino acid profiles between groups 2.5.1 Significant differences were found in serum amino acid profiles among the groups. From the PCA score chart ( Figure 2 (A) and PLS-DA score chart ( Figure 2 B) shows a significant trend of separation among the groups. Random permutation test ( Figure 2 As shown in C), the actual accuracy rate is 0.814. P The value <0.001 indicates that the model has a low risk of overfitting and good predictive ability, suggesting that there are significant differences in serum amino acid levels between groups.

[0075] 2.5.2 Significant differences exist in serum amino acid profiles between exercised and non-exercised individuals. like Figure 3 As shown, the PCA score chart ( Figure 3 (A) and PLS-DA score chart ( Figure 3 B) shows a significant trend of separation between the exercise and non-exercise groups, according to the random permutation test ( Figure 3 As shown in C), the actual accuracy rate is 0.749. P A value <0.001 indicates a difference in serum amino acids between the exercise group and the non-exercise group, meaning that exercise induces differences in serum amino acids. 2.5.3 There were significant differences in serum amino acid profiles between fasting morning and postprandial evening meals. like Figure 4 As shown, the PCA score chart ( Figure 4 In the A) and PLS-DA score graph ( Figure 4 In both B) the results showed a clear trend of separation in serum amino acids between fasting morning and postprandial evening, according to the random permutation test. Figure 4 The results in (C) show that the actual accuracy is 0.867. P <0.001 indicates that serum amino acid levels vary depending on the time of day and dietary status.

[0076] 2.5.4 There are significant differences in serum amino acid profiles between men and women. like Figure 5 As shown, the PCA score chart ( Figure 5 (A) and PLS-DA score chart ( Figure 5 (B) shows a trend of segregation of serum amino acids between male and female subjects. Random permutation test ( Figure 5 As shown in C), the actual accuracy rate is 0.819. P <0.001 indicates a gender difference in serum amino acids.

[0077] 2.6 Effects of Exercise on Serum Amino Acids 2.6.1 Changes in serum amino acids before and after exercise As shown in Table 8, there was no statistically significant difference in serum amino acid levels before and after the experiment in the control group. P >0.05). Serum leucine and isoleucine levels decreased after exercise (morning and evening combined) in the exercise group. P <0.05%, and elevated levels of nine amino acids, including tyrosine, methionine, phenylalanine, histidine, tryptophan, γ-aminobutyric acid, citrulline, glutamic acid, and glycine. P <0.05).

[0078] Table 8 Serum amino acid levels in the exercise group and control group

[0079] 2.6.2 Changes in serum amino acids during morning fasting exercise As shown in Table 9, after fasting exercise in the morning, significant changes were observed in 8 serum amino acids in male subjects and 7 in female subjects. P <0.05. The levels of four amino acids—leucine, isoleucine, arginine, and valine—were significantly decreased in both men and women. P <0.05), both tyrosine and methionine were significantly increased ( P <0.05. Phenylalanine and histidine levels were significantly elevated only in men ( P <0.05%, glycine levels were significantly elevated only in women ( P <0.05).

[0080] Table 9. Serum amino acid levels after morning fasting exercise.

[0081] 2.6.3 Changes in serum amino acids after evening exercise As shown in Table 10, serum levels of 15 and 10 amino acids, respectively, were significantly altered in male and female subjects after exercise following an evening meal. P <0.05. Leucine and isoleucine levels were significantly decreased in both men and women. P <0.05%, serum levels of eight amino acids, including tyrosine, methionine, phenylalanine, histidine, tryptophan, lysine, γ-aminobutyric acid, and citrulline, were significantly elevated in both men and women. P <0.05); ornithine levels decreased significantly only in men ( P <0.05), glycine, glutamine, glutamate, and aspartate levels were significantly elevated only in men ( P <0.05).

[0082] Table 10 Serum Amino Acid Levels After Evening Exercise

[0083] like Figure 6 As shown, the serum amino acid levels in the exercise group after exercise in the morning (fasting) and evening (postprandial) showed five significant trends.

[0084] like Figure 6 and Figure 7 As shown, leucine and isoleucine levels decreased after exercise in the morning on an empty stomach and after a meal in the evening, and the trends were consistent in both men and women, suggesting that they were mainly driven by exercise and were not related to diet, time of day, or gender.

[0085] like Figure 6 and Figure 8 As shown, serum tyrosine, methionine, phenylalanine, and histidine levels were elevated or trending upward in both men and women after morning fasting exercise, and the increase was further amplified after evening exercise, demonstrating the cumulative effect of diet and exercise.

[0086] Figure 6 and Figure 9 As shown, arginine and valine levels decreased in both men and women after morning fasting exercise, but no significant changes were observed after evening post-dinner exercise, suggesting that dietary intake has a restorative effect on exercise expenditure.

[0087] Figure 6 and Figure 10 As shown, after morning fasting exercise, there were no significant changes in tryptophan, lysine, γ-aminobutyric acid, and citrulline levels in both men and women, but these levels significantly increased after evening exercise following a meal, indicating that these changes are more dependent on dietary intake.

[0088] like Figure 6 and Figure 11 As shown, glutamine, glutamate, and aspartate levels remained unchanged in men after morning fasting exercise, but significantly increased after postprandial exercise. In women, there were no changes after either fasting or postprandial exercise. Ornithine levels decreased only in men after evening postprandial exercise, indicating that ornithine changes are not related to dietary intake but may be related to the time of day and exhibit gender differences.

[0089] Glycine levels rise in men after an evening meal followed by exercise, but in women they rise in the morning after an empty stomach following exercise. This suggests an interaction between sex and diet in the changes in glutamine, glutamate, aspartic acid, ornithine, and glycine.

[0090] 2.7 Correlation analysis of amino acids with blood biochemistry, heart rate, and energy expenditure 2.7.1 Correlation analysis after morning fasting exercise like Figure 12As shown in A, after exercise, isoleucine was positively correlated with heart rate, valine was negatively correlated with TG, tyrosine and phenylalanine were both negatively correlated with HDL-C, methionine was positively correlated with energy consumption, and histidine was negatively correlated with blood urea nitrogen.

[0091] Figure 12 As shown in B, after fasting exercise in the morning, women showed negative correlations with tyrosine and energy consumption, methionine and blood glucose, and glycine and LDL-C.

[0092] 2.7.2 Correlation analysis after evening meal exercise like Figure 13 As shown in Figure A, after exercise in men, leucine and phenylalanine were positively correlated with heart rate, phenylalanine was negatively correlated with AST, glycine, lysine, and aspartic acid were all negatively correlated with TC, and glycine was negatively correlated with LDL-C; ornithine was positively correlated with TC, HDL-C, and LDL-C, and negatively correlated with energy consumption; γ-aminobutyric acid was positively correlated with uric acid, citrulline was negatively correlated with ALT, and aspartic acid was positively correlated with creatinine.

[0093] like Figure 13 As shown in B, after exercise in women, leucine, isoleucine, tyrosine, and phenylalanine were all negatively correlated with uric acid, isoleucine was negatively correlated with blood glucose, methionine was positively correlated with AST, γ-aminobutyric acid was negatively correlated with ALT, and citrulline was positively correlated with blood urea nitrogen.

[0094] Example 2 An amino acid supplement composition based on exercise time and gender stratification includes an amino acid supplement composition 1 suitable for men after morning fasting exercise, an amino acid supplement composition 2 suitable for women after morning fasting exercise, an amino acid supplement composition 3 suitable for men after evening post-meal exercise, and / or an amino acid supplement composition 4 suitable for women after evening post-meal exercise. The amino acid supplement composition 1 is composed of the following active amino acid ingredients: leucine, isoleucine, arginine and valine; based on the daily dosage, the hydrolyzed leucine content is 2 g, the hydrolyzed isoleucine content is 1 g, the hydrolyzed arginine content is 2 g, and the hydrolyzed valine content is 1.5 g. The amino acid supplement composition 2 is composed of the following active amino acid ingredients: leucine, isoleucine, arginine, valine, and glycine; based on the daily dosage, the hydrolyzed leucine content is 1 g, the hydrolyzed isoleucine content is 0.5 g, the hydrolyzed arginine content is 2 g, the hydrolyzed valine content is 1.5 g, and the hydrolyzed glycine content is 2 g. The amino acid supplement composition 3 is composed of the following active amino acid ingredients: leucine, isoleucine and ornithine; based on the daily dosage, the content of hydrolyzed leucine is 2 g, the content of hydrolyzed isoleucine is 1 g, and the content of hydrolyzed ornithine is 1 g.

[0095] The amino acid supplement composition 4 is composed of the following active amino acid ingredients: leucine and isoleucine; based on the daily dosage, the content of hydrolyzed leucine is 2 g and the content of hydrolyzed isoleucine is 1 g. The leucine in the above-mentioned amino acid supplement composition is derived from L-leucine; the isoleucine is derived from L-isoleucine; the arginine is derived from L-arginine hydrochloride (Examples 3 and 4 use L-arginine hydrochloride); the valine is derived from L-valine; the glycine is derived from glycine; and the ornithine is derived from L-ornithine hydrochloride.

[0096] Example 3 An amino acid supplement composition based on exercise time and gender stratification includes an amino acid supplement composition 1 suitable for men after morning fasting exercise, an amino acid supplement composition 2 suitable for women after morning fasting exercise, an amino acid supplement composition 3 suitable for men after evening post-meal exercise, and / or an amino acid supplement composition 4 suitable for women after evening post-meal exercise. The amino acid supplement composition 1 is composed of the following active amino acid ingredients: leucine, isoleucine, arginine and valine; based on the daily dosage, the hydrolyzed leucine content is 1.5 g, the hydrolyzed isoleucine content is 0.75 g, the hydrolyzed arginine content is 0.5 g, and the hydrolyzed valine content is 1 g. The amino acid supplement composition 2 is composed of the following active amino acid ingredients: leucine, isoleucine, arginine, valine, and glycine; based on the daily dosage, the hydrolyzed leucine content is 1.5 g, the hydrolyzed isoleucine content is 0.75 g, the hydrolyzed arginine content is 0.5 g, the hydrolyzed valine content is 1 g, and the hydrolyzed glycine content is 1 g. The amino acid supplement composition 3 is composed of the following active amino acid ingredients: leucine, isoleucine and ornithine; based on the daily dosage, the content of hydrolyzed leucine is 1.5 g, the content of hydrolyzed isoleucine is 0.75 g, and the content of hydrolyzed ornithine is 0.5 g.

[0097] The amino acid supplement composition 4 is composed of the following active amino acid ingredients: leucine and isoleucine; based on the daily dosage, the content of hydrolyzed leucine is 1.5 g and the content of hydrolyzed isoleucine is 0.75 g. The leucine in the above-mentioned amino acid supplement composition is derived from L-leucine; the isoleucine is derived from L-isoleucine; the arginine is derived from L-arginine hydrochloride; the valine is derived from L-valine; the glycine is derived from glycine; and the ornithine is derived from L-ornithine hydrochloride.

[0098] Example 4 An amino acid supplement composition based on exercise time and gender stratification includes an amino acid supplement composition 1 suitable for men after morning fasting exercise, an amino acid supplement composition 2 suitable for women after morning fasting exercise, an amino acid supplement composition 3 suitable for men after evening post-meal exercise, and / or an amino acid supplement composition 4 suitable for women after evening post-meal exercise. The amino acid supplement composition 1 is composed of the following active amino acid ingredients: leucine, isoleucine, arginine and valine; based on the daily dosage, the hydrolyzed leucine content is 3 g, the hydrolyzed isoleucine content is 1.5 g, the hydrolyzed arginine content is 2 g, and the hydrolyzed valine content is 1.5 g. The amino acid supplement composition 2 is composed of the following active amino acid ingredients: leucine, isoleucine, arginine, valine, and glycine; based on the daily dosage, the hydrolyzed leucine content is 3 g, the hydrolyzed isoleucine content is 1.5 g, the hydrolyzed arginine content is 2 g, the hydrolyzed valine content is 1.5 g, and the hydrolyzed glycine content is 3 g. The amino acid supplement composition 3 is composed of the following active amino acid ingredients: leucine, isoleucine and ornithine; based on the daily dosage, the content of hydrolyzed leucine is 3 g, the content of hydrolyzed isoleucine is 1.5 g, and the content of hydrolyzed ornithine is 2 g.

[0099] The amino acid supplement composition 4 is composed of the following active amino acid ingredients: leucine and isoleucine; based on the daily dosage, the content of hydrolyzed leucine is 3 g and the content of hydrolyzed isoleucine is 1.5 g. The leucine in the above-mentioned amino acid supplement composition is derived from L-leucine; the isoleucine is derived from L-isoleucine; the arginine is derived from L-arginine hydrochloride; the valine is derived from L-valine; the glycine is derived from glycine; and the ornithine is derived from L-ornithine hydrochloride.

[0100] Example 5 Ten male subjects took amino acid supplement composition 1 from Example 2 25 minutes after completing fasted exercise in the morning. The dosage, calculated based on the total weight of the active amino acid ingredients, was 1 g after exercise. This group was designated as the experimental group. A placebo group was also established. The 10 men in the placebo group had no significant differences in height, weight, or physique compared to the experimental group. The amount and content of their exercise were the same as in the experimental group; the only difference was that they took the same dose of glucose.

[0101] The results showed that, compared with the placebo group, the experimental group had a 22% faster heart rate recovery rate 60 minutes after exercise. P <0.05; the serum TG level in the experimental group 2 hours after exercise was 18.3% lower than that in the placebo group ( P <0.05); After taking the amino acid supplement composition 1 of the present invention, isoleucine recovered to 93.5% of baseline, while only 68.2% in the placebo group ( P <0.01).

[0102] Example 6 Ten female subjects took amino acid supplement composition 2 from Example 2 30 minutes after completing fasted exercise in the morning. The dosage, based on the total weight of the active amino acid ingredients, was 2 g after exercise. This group was designated as the experimental group. A placebo group was also included. The 10 women in the placebo group had no significant differences in height, weight, or body type compared to the experimental group. Their exercise volume and content were the same as the experimental group; the only difference was that they took the same dose of glucose.

[0103] The results showed that the energy expenditure efficiency (energy expenditure per unit heart rate) of the experimental group after exercise was 15.6% higher than that of the placebo group. P <0.05; 2 hours after exercise, the LDL-C level in the experimental group was 12.4% lower than that in the placebo group ( P <0.05); the glycine recovery rate in the experimental group reached 94.2%, while the glycine recovery rate in the placebo group was only 71.5% ( P <0.01).

[0104] Example 7 Thirty healthy men were recruited and randomly divided into three groups: Group III (formula III) of the amino acid supplementation composition described in Example 2, the standard BCAA group (the components administered in this group were the same as those in Group III of Example 2, except that ornithine was replaced with valine), and the placebo group (administered the same dose of glucose). Each group consisted of 10 men. Supplementation was administered 40 minutes after exercise following an evening meal. The dosage was 1 g based on the total weight of the active amino acid components.

[0105] The results are shown in Table 11. Formula III significantly reduced blood ammonia, blood urea nitrogen and total cholesterol (TC) levels, accelerated ornithine recovery and reduced fatigue, confirming the multiple functional benefits of ornithine supplementation during men's evening exercise.

[0106] Table 11 Detection results for different groups

[0107] Note: This indicates that compared to formulation group III, P <0.05.

[0108] Example 8 Twenty healthy women were recruited and randomly assigned to four groups (Formula IV) of the amino acid supplement composition described in Example 2 (n=10) and a placebo group (n=10, the placebo group received the same dose of glucose). Supplementation was administered 40 minutes after exercise following an evening meal, at a dose of 1.5 g based on the total weight of the active amino acid components. Serum uric acid (UA) was measured before exercise, immediately after exercise, 60 min after exercise, and 24 h after exercise.

[0109] The results showed that the serum UA level in the formulation IV group was 312.5 ± 25.3 μmol / L 60 min after exercise, which was significantly lower than that in the placebo group (358.2 ± 30.1 μmol / L). P <0.01); In Formula IV group, UA recovered to 96.2%±4.5% of pre-exercise levels 24 h after exercise, compared to 108.5%±5.8% in the placebo group. P <0.05); 60 min after exercise, the recovery rates of leucine and isoleucine were 92.1%±4.2% and 89.5%±4.8%, respectively, compared to 63.4%±5.5% and 60.2%±5.1% in the placebo group. P <0.01). This indicates that the amino acid supplement composition 4 of the present invention not only replenishes the consumption of branched-chain amino acids, but also significantly helps to reduce uric acid levels after exercise, providing dual benefits for female athletes.

[0110] Example 9 A method for post-exercise amino acid supplementation includes the following steps: Subjects were categorized into four groups based on their exercise time, whether they exercised on an empty stomach, and their gender: men exercising on an empty stomach in the morning, men exercising after a meal in the evening, women exercising on an empty stomach in the morning, and women exercising after a meal in the evening. Then, according to the subject type, the amino acid supplement composition described in Example 2 was matched. Men took amino acid supplement composition 1 after exercising on an empty stomach in the morning, women took amino acid supplement composition 2 after exercising on an empty stomach in the morning, men took amino acid supplement composition 3 after exercising after a meal in the evening, and women took amino acid supplement composition 4 after exercising after a meal in the evening. The above-mentioned amino acid supplements should be taken within 30 minutes after exercise, and the dosage is 1 g based on the total weight of the active amino acid ingredients.

[0111] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An amino acid supplement composition based on exercise time and gender stratification, characterized in that, The product comprises an amino acid supplement composition 1 suitable for men after morning exercise on an empty stomach, an amino acid supplement composition 2 suitable for women after morning exercise on an empty stomach, an amino acid supplement composition 3 suitable for men after evening exercise after a meal, and / or an amino acid supplement composition 4 suitable for women after evening exercise after a meal; wherein the amino acid supplement composition 1 is composed of the following active amino acid ingredients: leucine, isoleucine, arginine, and valine; wherein the amino acid supplement composition 2 is composed of the following active amino acid ingredients: leucine, isoleucine, arginine, valine, and glycine; wherein the amino acid supplement composition 3 is composed of the following active amino acid ingredients: leucine, isoleucine, and ornithine; and wherein the amino acid supplement composition 4 is composed of the following active amino acid ingredients: leucine and isoleucine.

2. The amino acid supplement composition according to claim 1, characterized in that, In the amino acid supplement composition 1, based on the daily dosage, there are 1.5-3 g of hydrolyzed leucine, 0.75-1.5 g of hydrolyzed isoleucine, 0.5-2 g of hydrolyzed arginine, and 0.75-1.5 g of hydrolyzed valine.

3. The amino acid supplement composition according to claim 1, characterized in that, The amino acid supplement composition 2 contains, based on the daily dosage, 1.5-3 g of hydrolyzed leucine, 0.75-1.5 g of hydrolyzed isoleucine, 0.5-2 g of hydrolyzed arginine, 0.75-1.5 g of hydrolyzed valine, and 1-3 g of hydrolyzed glycine.

4. The amino acid supplement composition according to claim 1, characterized in that, The amino acid supplement composition 3 contains, based on the daily dosage, 1.5-3 g of hydrolyzed leucine, 0.75-1.5 g of hydrolyzed isoleucine, and 0.5-2 g of hydrolyzed ornithine.

5. The amino acid supplement composition according to claim 1, characterized in that, The amino acid supplement composition 4 contains, based on the daily dosage, 1.5-3 g of hydrolyzed leucine and 0.75-1.5 g of hydrolyzed isoleucine.

6. The amino acid supplement composition according to any one of claims 1 to 5, characterized in that, The compound source of the leucine includes L-leucine; the compound source of the isoleucine includes L-isoleucine; the compound source of the arginine includes L-arginine, L-arginine hydrochloride, or L-arginine-aspartic acid; the compound source of the valine includes L-valine; the compound source of the glycine includes glycine; and the compound source of the ornithine includes L-ornithine hydrochloride.

7. The amino acid supplement composition according to claim 1, characterized in that, The dosage forms of the amino acid supplement composition include powder, granules, tablets, capsules, or liquids.

8. The use of the amino acid supplement composition according to any one of claims 1 to 6 in the preparation of products for post-exercise recovery, heart rate regulation, blood lipid regulation, blood glucose stabilization, ammonia metabolism regulation and / or uric acid management.

9. A method for amino acid supplementation after exercise, characterized in that, Includes the following steps: Subjects were categorized into four groups based on their exercise time, whether they exercised on an empty stomach, and their gender: men exercising on an empty stomach in the morning, men exercising after a meal in the evening, women exercising on an empty stomach in the morning, and women exercising after a meal in the evening. Then, according to the subject type, the amino acid supplement composition described in claim 1 was matched. Men took amino acid supplement composition 1 after exercising on an empty stomach in the morning, women took amino acid supplement composition 2 after exercising on an empty stomach in the morning, men took amino acid supplement composition 3 after exercising after a meal in the evening, and women took amino acid supplement composition 4 after exercising after a meal in the evening.

10. The method according to claim 9, characterized in that, Take the corresponding amino acid supplement within 30 minutes after exercise.

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