A composition for improving muscle attenuation and promoting muscle growth and uses thereof

Abstract

This invention belongs to the field of functional composition technology, and specifically relates to a composition for improving muscle loss and promoting muscle growth, and its application. The composition comprises leucine, lupin peptide, lactoferrin, and broccoli peptide, wherein the mass ratio of leucine, lupin peptide, lactoferrin, and broccoli peptide is (1~5):(1~5):(1~10):(1~10). It has been demonstrated that the combined intervention of these four specific components has a synergistic effect in improving muscle loss, promoting muscle growth, enhancing muscle performance, and regulating the expression of muscle metabolism-related factors, showing broad application prospects in improving muscle condition and function.

Description

Technical Field

[0001] This invention belongs to the field of functional composition technology, and specifically relates to a composition for improving muscle loss and promoting muscle growth, and its application. Background Technology

[0002] Sarcopenia, also known as muscle wasting disease, is a progressive syndrome of skeletal muscle mass and function decline closely related to aging. With the accelerating aging of the global population, the incidence of this disease continues to rise, becoming a significant public health problem threatening the health and quality of life of the elderly. The occurrence of sarcopenia is usually associated with multiple factors, including insufficient physical activity, unbalanced nutritional intake, and chronic diseases (such as chronic inflammation). Patients clinically present with reduced muscle mass, decreased muscle strength, and limited mobility, thereby increasing the risk of falls and fractures, and in severe cases, loss of independent living ability. Furthermore, this disease also negatively impacts mental health and social participation. Therefore, developing safe and effective interventions for sarcopenia has significant clinical and social value.

[0003] Currently, intervention strategies for sarcopenia mainly include two directions: exercise intervention and nutritional supplementation. Regarding nutritional intervention, studies have confirmed that various active ingredients have regulatory effects on muscle metabolism. Leucine, as a key branched-chain amino acid, can precisely target and regulate the metabolic pathways of muscle protein synthesis and degradation, efficiently activate the mTOR signaling pathway, directly drive muscle protein synthesis, and simultaneously inhibit excessive muscle protein degradation, maintaining muscle mass and functional homeostasis. Lactoferrin is widely found in mammalian milk and is a core immune protein in breast milk, possessing various biological activities, including antiviral infection, antioxidant, and regulation of the body's immune response. Lactoferrin has both immunomodulatory and cellular protective effects; it can reduce muscle tissue inflammation, alleviate the erosive damage of chronic inflammation to muscles, protect mitochondrial function in muscle cells, inhibit abnormal apoptosis of muscle cells, and break the vicious cycle of muscle aging and functional decline.

[0004] Existing technologies attempt to combine food-derived substances with anti-inflammatory and antioxidant activities with high-quality protein in order to develop more effective compositions for improving muscle loss and promoting myogenic differentiation. However, the nutritional support and functional intervention effects of such products still need improvement. Currently, there are no reports on using lupin and broccoli protein peptides in combination with leucine and lactoferrin to improve muscle loss. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention aims to propose a composition that improves muscle loss and promotes muscle growth. By combining lupin protein peptides, broccoli protein peptides, leucine, and lactoferrin, it solves problems such as muscle atrophy, reduced muscle mass, and decreased muscle performance.

[0006] In a first aspect, the present invention provides a composition for improving muscle loss and promoting myoblast differentiation, the composition comprising leucine, lupin peptide, lactoferrin and broccoli peptide, wherein the mass ratio of leucine, lupin peptide, lactoferrin and broccoli peptide is (1~5):(1~5):(1~10):(1~10).

[0007] Lupin protein peptides are plant-derived storage globulins extracted from lupin seeds. They have a balanced amino acid composition and are rich in branched-chain amino acids, primarily participating in the synthesis and metabolism of proteins in the body, providing crucial nutritional support for muscle tissue repair and growth. Broccoli protein peptides are rich in various amino acids and active fragments, exhibiting significant antioxidant and anti-inflammatory activities. They can effectively scavenge excess free radicals generated by oxidative stress, reducing the damage of inflammatory responses to muscle cells. Furthermore, they can regulate cell signaling pathways, providing a favorable microenvironment for muscle cell proliferation and survival, and playing a positive role in maintaining the structural integrity of muscle tissue.

[0008] Furthermore, the mass ratio of leucine, lupin protein peptide, lactoferrin and broccoli protein peptide is (1~3):(1~3):(1~5):(1~5). Furthermore, the mass ratio of leucine, lupin peptide, lactoferrin, and broccoli peptide is 1:2:2-4:3.

[0009] In a second aspect, the present invention provides a product comprising the composition described in the first aspect.

[0010] Preferably, the product is feed, health food, or medicine, and the product further contains excipients acceptable for use in feed, health food, or medicine.

[0011] Thirdly, the present invention provides the use of the composition described in the first aspect in the preparation of products for improving muscle mass, muscle index or muscle performance; preferably, improving muscle performance includes improving muscle strength or muscle endurance.

[0012] Fourthly, the present invention provides the use of the composition described in the first aspect in the preparation of a product for promoting muscle growth.

[0013] Fifthly, the present invention provides the use of the composition described in the first aspect in the preparation of a product for delaying muscle atrophy.

[0014] In a sixth aspect, the present invention provides the use of the composition described in the first aspect in a product that regulates the expression of muscle metabolism-related factors; preferably, the muscle metabolism-related factors include muscle differentiation-related factors and muscle atrophy-related factors; more preferably, the muscle differentiation-related factors include Myod1, Myog, and Myf5, and the muscle atrophy-related factors include Foxo3 and Murf.

[0015] Compared with the prior art, the present invention has the following advantages: (1) This invention is the first to combine four active ingredients, namely leucine, lupin protein peptide, lactoferrin and broccoli protein peptide, and confirms that the combined intervention can promote muscle growth, delay muscle atrophy, enhance muscle strength and exercise ability, regulate the expression of muscle metabolism-related factors, and the four components have a synergistic effect, which has broad application prospects in improving muscle loss.

[0016] (2) As a preferred embodiment, the present invention further determined the specific ratio of the four components, and confirmed that the improvement indicators under this ratio are better than those under other ratios, and can better give full play to the synergistic effect of the four components, effectively improve muscle loss and promote muscle growth.

[0017] (3) The leucine, lupin protein peptide, lactoferrin and broccoli protein peptide used in this invention are all natural, safe and have no obvious toxic side effects. They are suitable for long-term oral intake. Compared with traditional drug intervention, they have higher compliance and are applicable to a wider range of people, especially the elderly, postoperative rehabilitation and sports nutrition groups.

[0018] (4) The raw materials of the composition of the present invention are readily available and the cost is controllable. It can be made into various dosage forms such as powder, granules, tablets, and oral liquids, which are easy to industrialize and market. It has potential industrialization advantages in the fields of functional foods, special medical purpose formula foods and dietary supplements. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the mouse animal experiment grouping and dosing regimen in an embodiment of the present invention; Figure 2 The changes in body weight of mice in each group during the intervention period; Figure 3 The mass of the gastrocnemius muscle in each group of mice; Figure 4 The gastrocnemius muscle index of mice in each group; Figure 5 The figures show the lean meat and fat content of mice in each group. In the figure, A represents the lean meat content of mice in each group; and B represents the fat content of mice in each group. Figure 6 The results of the suspension test for each group of mice are shown below; Figure 7 The grip strength of mice in each group; Figure 8 The time to exhaustion for mice in each group on the treadmill; Figure 9 The results of H&E staining of skeletal muscle fiber tissues of mice in each group are shown. Among them, A is the normal group, B is the model group, C is the leucine group, and D is the lupin protein peptide group. Figure 10 The results of H&E staining of skeletal muscle fiber tissues of mice in each group are shown. Among them, E is the lactoferrin group, F is the broccoli protein peptide group, G is the combined group 1, H is the combined group 2, and I is the combined group 3. Figure 11 The expression results of myoblast differentiation-related factors Myod1, Myog, and Myf5 in each group of mice; Figure 12 The mRNA expression levels of muscle atrophy-related factors Foxo3, Murf, and inflammation-related factor Icam1 in each group of mice were statistically analyzed.

[0020] Note: In the figure, * indicates a significant difference between groups (P<0.05), ** indicates a significant difference between groups (P<0.01), *** indicates a significant difference between groups (P<0.001), and **** indicates a significant difference between groups (P<0.0001). Detailed Implementation

[0021] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0022] A mouse model of sarcopenia was established using D-galactose (D-gal). Leucine, lupin peptide, lactoferrin, and broccoli peptide were administered individually or in combination to investigate the effects of different interventions on muscle movement, muscle synthesis, and muscle fiber atrophy. The experimental method involved intraperitoneal injection of galactose (D-gal) at a BW dose of 500 mg / kg / day to establish the sarcopenia model. Leucine, lupin peptide, lactoferrin, and broccoli peptide, as well as combinations of these peptides in different ratios, were administered via gavage. Mice in each group received the medication once daily via intraperitoneal and gavage for 56 days. During the experiment, mice had free access to food and water, and their weight was measured weekly.

[0023] I. Experimental Preparation Six-week-old male SPF-grade C57BL / 6 mice, weighing 20±1g each, were purchased from Sperry. They were housed at room temperature of 22±2℃ with a 12-hour light-dark cycle, and were allowed to acclimatize for one week after purchase. They were then randomly divided into 10 groups of 6 mice each. II. Experimental Grouping (1) Normal group (CON): injected with physiological saline and administered physiological saline by gavage; (2) Model group (MOD): The sarcopenia model was established by intraperitoneal injection of D-galactose at a dose of 500 mg / kg / day BW and by gavage with normal saline. (3) Leucine group (Leu group): Sarcopenia model was established by intraperitoneal injection of galactose at a dose of 500 mg / kg / day BW, and leucine (500 mg / kg / day BW) was administered by gavage. (4) Lupin protein peptide group (LPP group): Sarcopenia model was established by intraperitoneal injection of galactose at a dose of 500 mg / kg / day BW, and lupin protein peptide (500 mg / kg / day BW) was administered by gavage. (5) Lactoferrin group (Lf group): Sarcopenia model was established by intraperitoneal injection of galactose at a dose of 500 mg / kg / day BW, and lactoferrin (500 mg / kg / day BW) was administered by gavage. (6) Broccoli protein peptide group (BDP group): Sarcopenia model was established by intraperitoneal injection of galactose at a dose of 500 mg / kg / day BW, and broccoli protein peptide (500 mg / kg / day BW) was administered by gavage. (7) Combination Group 1: A sarcopenic model was constructed by intraperitoneal injection of galactose at a dose of 500 mg / kg / day BW. Combination Group 1 (500 mg / kg / day BW) was administered by gavage. Combination Group 1 was prepared by mixing leucine, lupin protein peptide, lactoferrin and broccoli protein peptide in a mass ratio of 1:2:2:3. (8) Combination Group 2: A sarcopenic model was constructed by intraperitoneal injection of galactose at a dose of 500 mg / kg / day BW. Combination Group 2 (500 mg / kg / day BW) was administered by gavage. Combination Group 2 was prepared by mixing leucine, lupin protein peptide, lactoferrin and broccoli protein peptide in a mass ratio of 1:2:3:3. (9) Combination Group 3: A sarcopenic model was constructed by intraperitoneal injection of galactose at a dose of 500 mg / kg / day BW. Combination Group 3 (500 mg / kg / day BW) was administered by gavage. Combination Group 3 was prepared by mixing leucine, lupin protein peptide, lactoferrin and broccoli protein peptide in a mass ratio of 1:2:4:3.

[0024] III. Experimental Methods 1. Weight monitoring The day after the last oral administration of the drug to the mice, a behavioral experiment was conducted. After the test, skeletal muscles such as the gastrocnemius muscle (GAS) were taken and weighed. The gastrocnemius muscle of the left leg of the mouse was preserved in tissue fixative for later use, while the gastrocnemius muscle of the right leg was flash-frozen in liquid nitrogen and placed in a freezer at -80°C for later use.

[0025] 2. Body composition analysis and imaging After the intervention in mice, the body composition of the mice was measured using a conscious small animal body composition analysis and imaging system (animal fat analyzer). Before testing, the mice were weighed and then placed in the self-tested instrument. After measurement, physiological parameters such as lean meat content and fat content in the conscious state of the mice were obtained. Each mouse was measured 2-3 times.

[0026] 3. Behavioral experiments a. Suspension experiment Mice were suspended on an inverted grid 60 cm high, with a thick mat placed underneath. Each mouse was placed in the center of the grid, which was then gently flipped upside down, with the mouse's head down. The suspension time was recorded until the mouse fell. Each mouse was tested twice, with an interval of >30 minutes between tests. The suspension time was recorded, and each mouse was scored according to a scoring criterion. If a mouse fell in less than 10 seconds, the test was repeated immediately to ensure the accuracy of the results. This test is based on the mouse's instinctive fear of falling; therefore, it is best to allow the mouse to complete the test under unknown conditions in a limited number of repetitions.

[0027] The scoring criteria are as follows: points are assigned based on the actual hanging time, with a maximum score of 100 points. For example, 120 seconds is scored as 12 points, and >1000 seconds is scored as the maximum score of 100 points.

[0028] b. Grip strength test The basic process of the grip strength measurement experiment consists of the following steps: (1) Install the elastic metal strip onto the sensor and ensure it is secure.

[0029] (2) Turn on the sensor and select peak mode. Do not apply force that exceeds the sensor’s strength tolerance range.

[0030] (3) Display sensor reset to zero.

[0031] (4) Take the mouse out of the cage and grasp the middle part of the mouse's tail with your thumb and forefinger.

[0032] (5) Forelimb Measurement: Place the mouse on the elastic metal strip, allowing its forepaws to contact the strip before measurement. Keep the torso and the strip horizontal. Gently pull the mouse's tail from the top of the strip until the mouse can no longer maintain its grip and the strip slips from its paws. During the test, the measuring device records the maximum grip strength and displays it on the instrument's screen. Repeat 3 times.

[0033] c. Treadmill test (1) Adaptation phase Table 1. Treadmill parameters during the adaptation phase The treadmill parameters for the adaptation phase are shown in Table 1. The treadmill stimulation current was set to 0.5 mA. This gradual training method was used to allow the animals to adapt to the treadmill. This adaptation process lasted 2-3 days, once a day, for approximately 10 minutes each time.

[0034] (2) Testing Phase Table 2. Treadmill parameters during the testing phase The treadmill parameters were set as shown in Table 2 during the testing phase. The stimulation current of the treadmill was set to 0.5 mA. Exhaustion was defined as the mouse stopping running for 10 seconds, followed by an additional 10 seconds of stimulation with electricity or noise, after which the mouse was still unable to run. The time to exhaustion was recorded. This process was repeated once daily for 2-3 days.

[0035] 4. Evaluation of pathological indicators Muscle slice preparation: Dehydration and paraffin infiltration: The GAS was removed from the tissue fixative and trimmed, placed in an embedding cassette and labeled. The cassette was then rinsed in running water for 2 hours. After rinsing, the embedding cassettes were neatly arranged and placed in a fully automated biological dehydrator. The automatic dehydration program was set, and the cassettes were removed after 12 hours of dehydration.

[0036] Tissue embedding: First, pour molten paraffin into the mold, carefully remove the tissue with heated tweezers, and quickly place it into the mold with the cross-section facing upwards. After the paraffin has slightly solidified, cover it with the embedding box base, continue to add wax until the embedding box base is submerged, and allow it to cool to room temperature and solidify.

[0037] Sectioning and spreading: Remove the embedded wax block from the mold and place it on the biological tissue freezing stage. After the wax block cools, fix it on the paraffin microtome. First, adjust the section thickness and trim the section. When complete muscle tissue appears, start continuous sectioning. Each section is 5 μm thick. Use tweezers to remove the section and spread it flat on the water surface of the slide spreader. Use curved tweezers to unfold the folds. The spreading temperature is 42℃.

[0038] Slicing and baking: Once the slices are fully expanded, quickly lift them out with a glass slide, absorb excess water with filter paper, write the numbers on them, and place them on a baking machine. Bake the slices at 42℃ for 2 hours. After baking, place the slices in a 60-70℃ oven overnight to prevent them from falling off.

[0039] At this point, the sections can be dewaxed and rehydrated. The system is shown in Table 3: Table 3 Dewaxing and Rehydration System Then, pathological H&E staining was performed.

[0040] ① First, immerse the glass slide in the hematoxylin staining solution for 10 minutes; ②Then rinse with water for 5-10 minutes; ③ Then stain in 1% eosin solution for 30s~1min; ④ Ethanol dehydration; ⑤ Finally, seal the film with neutral resin.

[0041] 5. Detection of expression levels of muscle-building-related genes Take a portion of muscle tissue, add 1 ml of Trizol to a grinding tube, and grind it using a tissue homogenizer at 8000 rpm for 30 seconds, repeating this process 3 times. After each grinding, incubate on ice for 1 minute to extract tissue RNA. ① Add 200 μl of chloroform to the ground tube and centrifuge at 12000 r / min for 15 min at 4℃.

[0042] ② Transfer 500 μl of supernatant into an enzyme-free 1.5 ml EP tube containing an equal volume of pre-cooled isopropanol and let stand for 10 min.

[0043] ③ Centrifuge at 12000 r / min for 10 min in a 4℃ centrifuge.

[0044] ④ Discard the supernatant, add 75% ethanol prepared with pre-cooled DEPC water, and centrifuge at 12000 r / min for 5 min at 4℃.

[0045] ⑤ Repeat step ④.

[0046] ⑥ Discard the supernatant, air dry at room temperature for 30 min, add DEPC water at 4℃ to dissolve the RNA, and measure the concentration.

[0047] The obtained tissue RNA was reverse transcribed, and the amplification system is shown in Table 4: Table 4 Reverse Transcription Amplification System The internal reference gene was β-actin, and the levels of myogenic differentiation factor 1 (Myod1), myopoietin (Myog), myocyte enhancement factor 2C (Mef2c), myogenic factor 5 (Myf5), forkhead box protein O3 (Foxo3), muscle ring finger protein (Murf), and intercellular adhesion molecule 1 (Icam1) were analyzed.

[0048] The real-time PCR system is shown in Table 5: Table 5 Real-time PCR System Performance Test Example 1: Effects of Different Compositions on Improving Muscular Atrophy in Mice like Figure 3-10 As shown, compared with the normal group, the model group showed a significant decrease in grip strength, time to exhaustion in the treadmill test, gastrocnemius muscle mass, and cross-sectional area of ​​skeletal muscle fibers, indicating that the mouse muscle atrophy model was successfully established.

[0049] from Figure 2 It can be seen that the weight of mice in each group increased during the intervention process, and the trends were similar, with little difference in weight among the groups.

[0050] Depend on Figure 3 and Figure 4 It can be seen that, from Figure 3 and Figure 4 It can be seen that, compared with the normal group (CON group) mice, the muscle weight of the hind limb gastrocnemius muscle in the model group (MOD group) mice was significantly reduced. The gastrocnemius muscle and muscle index are criteria for judging muscle atrophy; the significantly reduced muscle index of the hind limb gastrocnemius muscle indicates that the aging group mice developed age-related muscle atrophy, and the experimental model was successfully established. Among other things: Compared to the model group (MOD group) mice, the combined intervention with leucine, lupin peptide, lactoferrin, and broccoli peptide significantly increased the muscle weight and muscle index of the hind limb gastrocnemius muscle in combined groups 1, 2, and 3, and was significantly higher than that in the Leu group, LPP group, Lf group, and BDP group at the same dose. This indicates that the muscle mass of the mice after combined intervention was significantly better than that of the single-component intervention groups. It can be seen that leucine, lupin peptide, lactoferrin, and broccoli peptide have a synergistic effect in improving muscle mass and muscle percentage. Further comparison of combined groups 1, 2, and 3 showed that all three groups improved the muscle weight and muscle index of the gastrocnemius muscle in aging mice to varying degrees. However, the improvement effect of combined group 3 was the most significant, with the muscle weight and muscle index of the gastrocnemius muscle being higher than those of combined groups 1 and 2. This indicates that among the three ratios set in this experiment, combined group 3 (with a mass ratio of leucine, lupin peptide, lactoferrin, and broccoli peptide of 1:2:4:3) had the best effect on improving age-related muscle atrophy.

[0051] Performance Test Example 2: Effects of Different Compositions on Improving Body Composition in Mice The increased lean meat content in mice indicates an increase in the proportion of muscle tissue and improved skeletal muscle development and growth; the decrease in fat content combined with the increase in lean meat content suggests that the combined intervention can effectively reverse this imbalance and restore a healthy body composition ratio.

[0052] from Figure 5 As shown in A and B, compared with the model group (MOD group) mice, the combined intervention of leucine, lupin peptide, lactoferrin, and broccoli peptide significantly increased the lean meat content of mice in the combined group 3, which was significantly higher than that of the Leu group, LPP group, Lf group, and BDP group at the same dose. The fat content of combined groups 1, 2, and 3 was significantly reduced, which was significantly lower than that of the Leu group, LPP group, Lf group, and BDP group at the same dose. This indicates that the combined intervention had a significantly better effect on improving body composition than the single-component intervention groups. It can be seen that leucine, lupin peptide, lactoferrin, and broccoli peptide have a synergistic effect in improving body composition. Further comparison of combined groups 1, 2, and 3 shows that there are significant differences in the improvement of body composition among combined groups 1, 2, and 3. Among them, combined group 3 showed the most significant increase in lean meat content and the most significant decrease in fat content, which was better than combined groups 1 and 2.

[0053] The above results indicate that combined intervention can effectively reverse the body composition imbalance caused by aging and restore a healthy body composition ratio. Under the conditions of this experiment, combined group 3 (the mass ratio of leucine, lupin peptide, lactoferrin and broccoli peptide was 1:2:4:3) had the best regulatory effect on body composition.

[0054] Performance Test Example 3: Effects of Different Compositions on Muscle Motion Ability in Mice from Figure 6 , Figure 7 It can be seen that, compared with the model group (MOD group), the combined intervention of leucine, lupin peptide, lactoferrin, and broccoli peptide significantly improved the grid suspension time and grip strength of mice in the combined group 3, and the effect was significantly higher than that of the Leu group, LPP group, Lf group, and BDP group at the same dose, indicating that the combined intervention can better enhance the muscle strength of mice. The grid suspension time and grip strength of mice after the combined intervention were significantly better than those of the single-component intervention groups, indicating that leucine, lupin peptide, lactoferrin, and broccoli peptide have a synergistic effect in improving muscle motor ability. Further comparison of combined group 1, combined group 2, and combined group 3 shows that there are significant differences in the enhancement effect of combined group 1, combined group 2, and combined group 3 on the muscle strength of mice. Among them, the combined group 3 mice had the longest grid suspension time and the greatest grip strength, and its improvement effect was significantly better than that of combined group 1 and combined group 2. The above results indicate that the formulation of combination group 3 has the best effect on enhancing muscle strength and endurance in mice, effectively improving muscle movement ability and helping to improve age-related muscle function.

[0055] from Figure 8 It can be seen that, compared with the model group (MOD group), the combined intervention of leucine, lupin peptide, lactoferrin, and broccoli peptide significantly prolonged the time to exhaustion in the treadmill test for mice in combined groups 1, 2, and 3, and the effect was significantly higher than that of the Leu group, LPP group, Lf group, and BDP group at the same dose. This indicates that the time to exhaustion in the treadmill test of mice after combined intervention was significantly longer than that of the single-component intervention groups. It can be seen that leucine, lupin peptide, lactoferrin, and broccoli peptide have a synergistic effect in improving the muscle movement ability of mice. Further comparison of combined groups 1, 2, and 3 shows that there are significant differences in the improvement of muscle function in mice. Among them, the time to exhaustion in the treadmill test of mice in combined group 3 was the longest, and its prolongation effect was significantly better than that in combined groups 1 and 2. The above results indicate that combined interventions with different ratios can improve muscle motor ability in aging mice. Among the three ratios set in this experiment, combined group 3 (leucine, lupin peptide, lactoferrin and broccoli peptide in a mass ratio of 1:2:4:3) showed the best effect on improving muscle motor ability in mice and was more conducive to alleviating age-related muscle function decline and improving muscle function.

[0056] Performance Test Example 4: Effects of Different Compositions on Muscle Loss in Mice from Figure 9 , Figure 10The H&E staining results of mice in each group showed that in the normal group (CON group), skeletal muscle fibers were tightly arranged, uniform in diameter, and exhibited polygonal cross-sections with cell nuclei located at the cell edges. In the model group (MOD group), skeletal muscle fiber structure was locally loose, disordered, and the spacing between muscle fibers was widened. Compared with the model group (MOD group), mice in combined groups 1, 2, and 3 showed increased cross-sectional area of ​​muscle fibers, more regular cell arrangement, and reduced interstitial components, with significantly better results than the Leu, LPP, Lf, and BDP groups at the same dosage. The results indicate that the morphology of skeletal muscle fibers in mice after combined intervention was significantly better than that of the single-component intervention groups. This suggests that leucine, lupin peptide, lactoferrin, and broccoli peptide have a synergistic effect in improving the morphology of skeletal muscle fibers in mice. Further comparison of combined groups 1, 2, and 3 reveals significant differences in their effects on sarcopenia. Combined group 3 showed the most tightly packed muscle fibers, the largest cross-sectional area, the least interstitial component, and the muscle fiber morphology closest to the normal group (CON group), demonstrating a significantly better improvement effect than combined groups 1 and 2. This indicates that combined interventions with different ratios are more effective in improving sarcopenia than interventions with leucine, lupin peptide, lactoferrin, or broccoli peptide alone. Furthermore, among the three ratios used in this experiment, combined group 3 (with a mass ratio of leucine, lupin peptide, lactoferrin, and broccoli peptide of 1:2:4:3) showed the best effect in improving sarcopenia.

[0057] Performance Test Example 5: Effects of Different Compositions on Myogenic Differentiation-Related Factors and Muscle Atrophy-Related Factors in Mice from Figure 11 , Figure 12The expression of myogenic differentiation-related factors in each group of mice showed that, compared with the normal control group (CON group), the mRNA expression of myogenic differentiation-related factors Myod1, Myog, and Myf5 in the model group (MOD group) was significantly decreased, indicating that the expression of myogenic differentiation-related factors decreases and the differentiation capacity of myoblasts declines in aging. After 8 weeks of intervention, compared with the model group (MOD group), the mRNA expression of myogenic differentiation-related factors in combined groups 1, 2, and 3 was increased, and the differentiation capacity was improved. Moreover, compared with the Leu group, LPP group, Lf group, and BDP group, combined groups 1 and 2 showed significantly higher expression of myogenic differentiation-related factors. The mRNA expression of myoblast differentiation-related factors in combined group 3 was superior to that in the single-component groups at the same dosage, indicating that the mRNA expression of myoblast differentiation-related factors in mice after combined intervention was significantly better than that in the single-component intervention groups. This shows that leucine, lupin peptide, lactoferrin, and broccoli peptide have a synergistic effect in improving the mRNA expression of myoblast differentiation-related factors in mice. Further comparison of combined groups 1, 2, and 3 shows that there are significant differences in the enhancement of myoblast differentiation-related factor expression among combined groups 1, 2, and 3. Among them, the mRNA expression levels of myoblast differentiation-related factors Myod1, Myog, and Myf5 in combined group 3 were the highest and closest to the normal control group (CON group). The synergistic effect of combined group 3 (the mass ratio of leucine, lupin peptide, lactoferrin, and broccoli peptide was 1:2:4:3) was the best, which can more effectively reverse the decline in myoblast differentiation function caused by aging and enhance the differentiation capacity of myoblasts.

[0058] Compared with the normal control group (CON group), the mRNA expression of muscle atrophy-related factors Foxo3 and Murf in the model group (MOD group) was significantly increased, indicating that in the aging state, the expression of muscle atrophy-related factors increases, muscle fiber degradation intensifies, and muscle function declines. After 8 weeks of combined intervention experiments with different ratios of leucine, lupin peptide, lactoferrin, and broccoli peptide, the mRNA expression of muscle atrophy-related factors Foxo3 and Murf in combined groups 1, 2, and 3 was decreased compared with the model group (MOD group), muscle fiber degradation was inhibited, and muscle function was improved. Moreover, compared with the Leu group, LPP group, Lf group, and BDP group, the degree of decrease in the mRNA expression of muscle atrophy-related factors Foxo3 and Murf in each combined intervention group was better than that of the single component groups at the same dose, indicating that the combined intervention reduced muscle atrophy-related factors in mice. The decrease in mRNA expression of the factors was significantly better than that of the single-component intervention group, indicating that leucine, lupin peptide, lactoferrin, and broccoli peptide have a synergistic effect in reducing the mRNA expression of muscle atrophy-related factors in mice. Further comparison of the combined intervention groups with different ratios showed that the downregulation effects of combined group 1, combined group 2, and combined group 3 on the expression of muscle atrophy-related factors were significantly different. Among them, the mRNA expression levels of muscle atrophy-related factors Foxo3 and Murf in combined group 3 were the lowest, closest to the normal control group (CON group), and its decrease was significantly better than that in combined group 1 and combined group 2. This indicates that the intervention effect of combined group 3 (the mass ratio of leucine, lupin peptide, lactoferrin, and broccoli peptide was 1:2:4:3) was the best, which can more effectively improve the process of age-related muscle atrophy and has a better intervention effect on maintaining muscle homeostasis and delaying muscle function decline.

[0059] Compared with the normal control group (CON group), the mRNA expression of the inflammation-related factor Icam1 in the model group (MOD group) was significantly increased, indicating that the expression of the inflammation-related factor Icam1 increases in the aging state, and the body's inflammatory response is enhanced. After 8 weeks of intervention, compared with the model group (MOD group), the mRNA expression of the inflammation-related factor Icam1 in combined groups 1, 2, and 3 decreased, and the inflammatory response was weakened. Moreover, compared with the Leu group, LPP group, Lf group, and BDP group, the degree of decrease in the mRNA expression of the inflammation-related factor Icam1 in combined groups 1, 2, and 3 was better than that in the single-component groups at the same dosage, indicating that the degree of decrease in the mRNA expression of the inflammatory factor in mice after combined intervention was significantly better than that in the single-component intervention groups. It can be seen that leucine, lupin peptide, lactoferrin, and broccoli peptide have a synergistic effect in improving the degree of decrease in the mRNA expression of the inflammatory factor in mice. Further comparison of combined groups 1 and 2... Compared with group 3, there were significant differences in the downregulation effects of group 1, group 2, and group 3 on the expression of inflammation-related factors. Among them, group 3 had the lowest mRNA expression level of the inflammation-related factor Icam1, which was closest to the normal control group (CON group), and its degree of reduction was significantly better than that of group 1 and group 2. Group 3 (with a mass ratio of leucine, lupin protein peptide, lactoferrin, and broccoli protein peptide of 1:2:4:3) had the best anti-inflammatory effect, which could significantly reduce the expression of the aging-induced inflammation-related factor Icam1 mRNA, effectively alleviate the body's inflammatory response, and reverse the gene expression changes caused by aging, thereby delaying aging.

[0060] This invention is the first to combine four active ingredients—leucine, lupin peptide, lactoferrin, and broccoli peptide—and demonstrates that this combined intervention can promote muscle growth, delay muscle atrophy, enhance muscle strength and athletic ability, and regulate the expression of muscle metabolism-related factors. Furthermore, the four components exhibit a synergistic effect, showing broad application prospects in improving muscle loss. This invention further determines the optimal mass ratio of the four components to be 1:2:4:3. Under this ratio, all improvement indicators are superior to other combinations, fully leveraging the synergistic effect of the four components to effectively improve muscle loss.

[0061] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0062] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composition for improving muscle loss and promoting muscle growth, characterized in that, The composition comprises leucine, lupin peptide, lactoferrin and broccoli peptide, wherein the mass ratio of leucine, lupin peptide, lactoferrin and broccoli peptide is (1~5):(1~5):(1~10):(1~10).

2. The composition according to claim 1, characterized in that, The mass ratio of leucine, lupin peptide, lactoferrin and broccoli peptide is (1~3):(1~3):(1~5):(1~5).

3. The composition according to claim 1, characterized in that, The mass ratio of leucine, lupin peptide, lactoferrin, and broccoli peptide is 1:2:2-4:

3.

4. A product characterized in that, The product comprises the composition according to any one of claims 1-3.

5. The product according to claim 4, characterized in that, The product is feed, health food, or medicine, and the product also contains excipients acceptable for use in feed, health food, or medicine.

6. The use of the composition according to any one of claims 1-3 in the preparation of a product for improving muscle mass, muscle index or muscle performance; preferably, improving muscle performance includes improving muscle strength or muscle endurance.

7. Use of the composition according to any one of claims 1-3 in the preparation of a product for promoting muscle growth.

8. Use of the composition according to any one of claims 1-3 in the preparation of a product for delaying muscle atrophy.

9. The use of the composition according to any one of claims 1-3 in the preparation of a product for regulating the expression of muscle metabolism-related factors; preferably, the muscle metabolism-related factors include muscle differentiation-related factors and muscle atrophy-related factors.

10. The application according to claim 9, characterized in that, The muscle differentiation-related factors include Myod1, Myog, or Myf5, and the muscle atrophy-related factors include Foxo3 or Murf.

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