Polypeptide and use thereof for promoting muscle growth and improving exercise performance

Abstract

To provide a polypeptide and its use for promoting muscle growth and improving exercise performance.SOLUTION: The present invention discloses a polypeptide and its use for promoting muscle growth and improving exercise performance, since the polypeptide disclosed in the present invention has the activity of regulating the expression of proteins such as p-AMPK, AKT, and mTOR in the muscle growth pathway, by administering an effective amount of the polypeptide disclosed in the present invention or a composition containing the polypeptide to an individual, the muscle growth and muscle strength of the individual can be promoted, thereby achieving the effects of preventing or treating muscle damage-related diseases and improving exercise performance.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to small proteins and uses thereof, and more particularly to polypeptides and uses thereof for promoting muscle growth and improving athletic performance. [Background technology]

[0002] "Sarcopenia" refers to a disease in which the mass and function of whole-body skeletal muscle continues to decline. Patients with sarcopenia experience a decline in muscle mass, making them unable to stand or walk for long periods of time, unable to lift heavy objects, and more susceptible to falls, which affects their lifestyle and behavior. In severe cases, they may require care, become unable to live independently, and have an increased risk of death. Sarcopenia is more likely to occur in the elderly, but even younger people can suffer from it. The main causes are inadequate protein intake, malnutrition, lack of exercise habits, or an inappropriate diet.

[0003] Currently, there are no clinically available drugs that can treat sarcopenia, and patients can only be recommended nutritional supplements and exercise to increase muscle strength, but many patients avoid exercise due to lack of muscle strength or poor athletic performance. Many protein-rich nutritional foods have appeared on the market, claiming to be able to prevent sarcopenia, but protein supplementation cannot improve muscle mass or athletic performance; it only slows the loss of muscle mass and does not effectively treat or prevent sarcopenia or its complications. Summary of the Invention [Problem to be solved by the invention]

[0004] The main object of the present invention is to provide a polypeptide and its use for promoting muscle growth and improving athletic performance, that is, by daily supplementation with the polypeptide disclosed in the present invention, muscle mass can be effectively strengthened and muscle growth can be achieved, and the effects of preventing or treating diseases caused by muscle deficiency or muscle damage can be achieved.

[0005] Another object of the present invention is to provide a polypeptide and its use for promoting muscle growth and improving athletic performance, i.e., by administering the polypeptide disclosed in the present invention or a composition thereof to an individual, the explosive power and endurance of the individual's athletic performance can be improved. [Means for solving the problem]

[0006] To achieve the above-mentioned objectives, the present invention discloses a polypeptide and its use, wherein the amino acid sequence of the polypeptide includes a specific sequence or an amino acid sequence having 95% or more identity thereto, which is derived by substituting, deleting or adding one or more amino acids therein, wherein the specific sequence is SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 5.

[0007] In one embodiment of the present invention, the amino acid sequence of the polypeptide is any one of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5. [Effects of the Invention]

[0008] The polypeptide disclosed in the present invention has the activity of promoting muscle synthesis and growth, and therefore, by administering an effective amount of the polypeptide disclosed in the present invention or a composition containing the polypeptide to an individual, the muscle mass and strength of the individual can be effectively improved or enhanced.

[0009] Specifically, when an individual has symptoms of muscle mass deficiency, administering an effective amount of the polypeptide disclosed in the present invention or a composition containing the polypeptide to the individual can achieve the effect of treating and / or preventing diseases associated with muscle mass deficiency, such as sarcopenia, bone fractures, and osteoporosis.

[0010] In another embodiment of the present invention, the effect of improving the explosive power and endurance of an individual's exercise can be effectively achieved by administering an effective amount of the polypeptide disclosed in the present invention or a composition containing the polypeptide to an individual.

[0011] Additionally, the polypeptides disclosed herein can be derived from shellfish extracts, such as extracts of Mytilus edulis, and extracts of clams. [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows a comparison of chromatograms obtained after HPLC analysis of different seafood extracts and the polypeptides shown in SEQ ID Nos. 1 to 3 disclosed in the present invention. [Figure 2] 1 shows a comparison of chromatograms obtained after HPLC analysis of different seafood extracts and the polypeptides shown in SEQ ID Nos. 4 and 5 disclosed in the present invention. [Figure 3] This shows the results of a comparison of HPLC chromatograms of Mytilus mussel raw material, Mytilus mussel extract, and Mytilus mussel hydrolysate. [Figure 4] The total distance traveled by mice in each group was measured at 1 minute, 3 minutes, and 15 minutes. [Figure 5] The running speed of mice in each group was analyzed at 1 minute, 3 minutes, and 15 minutes. [Figure 6] The limb grip strength of mice in each group. [Figure 7] This is the total distance that mice in each group ran each day during the test period. [Figure 8] 1 shows the testosterone content in the serum of mice in each group. [Figure 9] This shows the results of an analysis of the change in muscle mass for each subject during the test period (14 days). [Figure 10] This is the total result of the grip strength test for both hands for each subject. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention discloses a polypeptide and its use for promoting muscle growth and improving athletic performance. The polypeptide disclosed in the present invention is a small molecule consisting of 9 to 12 amino acids and has the activity of controlling the expression of proteins such as p-AMPK, AKT, and mTOR in the muscle growth pathway. Therefore, by administering an effective amount of the polypeptide disclosed in the present invention or a composition containing the polypeptide to an individual, muscle growth and muscle strength in the individual can be promoted, thereby achieving the effects of preventing or treating diseases related to muscle damage and improving athletic performance.

[0014] Here, when the individual is a human, the effective amount of the composition is at least 300 to 600 mg / day.

[0015] In one embodiment of the present invention, the sequences of the polypeptides are those shown in SEQ ID No. 1 to SEQ ID No. 5, respectively.

[0016] In another embodiment of the present invention, the polypeptide sequence is a sequence that has 95% or more identity with any of the sequences set forth in SEQ ID No. 1 to SEQ ID No. 5, for example, the polypeptide sequence is a derivative obtained by substituting, adding, deleting, modifying, or combining any of the amino acids in any of the sequences set forth in SEQ ID No. 1 to SEQ ID No. 5.

[0017] In one embodiment of the present invention, the polypeptide may be prepared by artificial synthesis methods well known in the art or produced on a biological production platform. Specifically, a nucleic acid molecule that can be transcribed into the polypeptide by biotechnology well known in the art may be introduced into a recombinant vector, and the recombinant vector may then be introduced into a host cell for expression to obtain a substance containing the polypeptide, which may then be purified and separated.

[0018] In another embodiment of the present invention, the polypeptide disclosed herein may be derived from a shellfish extract, such as an extract of European mussel, which is obtained by extracting a specific shellfish with a specific extraction solvent, preferably solvent extraction, ultrasonic extraction, supercritical extraction, or a combination of at least two of the above methods, and the extraction solvent may be water, ethanol, methanol, or any other solution known in the art.

[0019] In another embodiment of the present invention, the polypeptides disclosed herein may be derived from shellfish hydrolysates, such as blue-green mussel hydrolysates, which are obtained by hydrolysis with a hydrolase, preferably an alkaline protease or other hydrolase known in the art and acceptable in the food industry.

[0020] In one embodiment of the invention, the composition comprises one of the polypeptides set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 5.

[0021] In another embodiment of the present invention, the composition comprises polypeptides set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5.

[0022] The term "European mussel" refers to a type of shellfish seafood also known as "mussel" or "mussel."

[0023] The term "Mussel powder" refers to a product obtained by thoroughly boiling mussels, removing the shells, drying, and crushing them.

[0024] The term "sarcopenia" refers to a condition characterized by a continuous decline in whole-body skeletal muscle mass and function. Clinically, sarcopenia is assessed based on factors such as an individual's ability to lift heavy objects, whether they need assistance walking, difficulty rising from a chair, number of stairs they can climb, and number of falls.

[0025] The term "comorbidities of sarcopenia" refers to diseases or conditions caused by sarcopenia, such as disability and physical weakness, osteoporosis, fractures, cognitive impairment, metabolic syndrome, and cardiovascular disease.

[0026] The term "effective amount" refers to the dosage required to prevent, ameliorate or treat the disease or condition being addressed.

[0027] The term "treatment" means that when a polypeptide disclosed in the present invention or a composition containing said polypeptide is administered to an individual, an improvement in the disease or symptom sought may be achieved or the progression of the disease sought may be delayed.

[0028] The term "prevention" means that when an individual is at risk of contracting a desired disease, the onset of the desired disease can be avoided or the timing of onset of the desired disease can be delayed by administering a polypeptide disclosed in the present invention or a composition containing said polypeptide to an individual.

[0029] The term "composition" refers to a composition containing a polypeptide or a substance containing the polypeptide disclosed in the present invention as a major active ingredient, and the composition can be manufactured into various product forms and modes depending on demand, such as dietary supplements, foods, medicines, beverages, etc. Furthermore, the composition can be prepared into various dosage forms, such as tablets, powder packets, jellies, etc., depending on factors such as the product form, administration target, and market sales.

[0030] In the following, several experimental examples will be given to explain the technical features and effects of the present invention.

[0031] The cells used in the following examples are all readily available to those skilled in the art, and therefore no patent deposit is required.

[0032] The animal experiments carried out in the following examples conformed to ethical guidelines regarding animal experiments.

[0033] The numerical values ​​of the operating conditions, data, dosages, etc. described in the following examples are merely illustrative of the present invention and do not limit the scope of protection or interpretation of the present invention. Those skilled in the art should understand, based on common knowledge or experimental practice, that the data or numerical values ​​disclosed in the following examples include an acceptable error range. That is, based on common knowledge in the technical field to which the present invention pertains, the acceptable error range is within ±10% of the disclosed numerical value. For example, the acceptable range for a temperature of 37°C is 37±3.7°C.

[0034] The dosages of the samples used in the following animal experiments are merely illustrative. That is, according to the general knowledge of the present invention, the dosages shown in the following examples may be converted according to the type of substance to be administered and the conditions under which it is administered, and the scope of protection of the present invention is not limited thereby.

[0035] Example 1: Preparation of synthetic sequences

[0036] The polypeptides shown in SEQ ID No. 1 to SEQ ID No. 5 were synthesized by artificial methods, and the sequences were confirmed to be error-free. The results are shown in Table 1.

[0037] Table 1: Information on each polypeptide JPEG2026019993000002.jpg47150

[0038] Example 2: Cellular testing

[0039] 1x10 C2C12 myoblasts 4The cells were seeded onto a 24-well plate at a concentration of 100 counts / mL and cultured in the following culture medium at 37°C in 5% CO2, with the culture medium being replaced every 2 to 3 days.

[0040] Culture medium: DMEM medium containing 10% fetal bovine serum. Antibiotics may be added. If antibiotics are added, the final concentrations of penicillin and streptomycin should be 10.0 IU / mL and 10.0 IU / mL, respectively.

[0041] When C2C12 myoblasts reached 90% confluency, the culture medium was replaced with DMEM medium containing 2% horse serum. The culture medium was replaced every 48 hours. After a total of four days of culture, if myotube differentiation and formation in the C2C12 myoblasts was observed, the subsequent grouping experiment could be carried out.

[0042] C2C12 myoblast cells were divided into groups and cultured under the following conditions.

[0043] The first group was a blank group.

[0044] The second group was a positive control group, to which 100 nM insulin was added.

[0045] The third group was a sample group, to which 400 μg / ml of sample was added, where the sample was the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 in Example 1.

[0046] After culturing the cells in each group for 24 hours, the cells were harvested and proteins were extracted. The expression levels of the following proteins were analyzed by Western blotting: AMPK, p-AMPK, mTOR, p-mTOR, AKT, and p-AKT. The relative expression folds compared to a blank group containing no sample were calculated. The results are shown in Table 2 below. The blank group had values ​​of 1 for p-AMPK / AMPK, p-AKT / AKT, and p-mTOR / mTOR.

[0047] Table 2: Protein analysis results for each polypeptide JPEG2026019993000003.jpg53149

[0048] As can be seen from the results in Table 2, each polypeptide disclosed in the present invention can have the activity of enhancing the expression of proteins such as p-AMPK, AKT, and mTOR. As is well known to those skilled in the art, p-AMPK, AKT, and mTOR are key factors that control muscle growth in the muscle growth mechanism. Therefore, the above results demonstrate that any of the polypeptides having the sequences shown in SEQ ID Nos. 1 to 5 disclosed in the present invention have the activity of enhancing the expression of p-AMPK, AKT, and mTOR. It was also shown that when an effective amount of any of the polypeptides having the sequences shown in SEQ ID Nos. 1 to 5 disclosed in the present invention is administered to an individual, it can achieve the effects of preventing or treating sarcopenia or its related symptoms, or improving exercise performance, by promoting muscle cell regeneration, degrading damaged muscle, and promoting muscle cell proliferation.

[0049] Example 3: Preparation of seafood extract

[0050] In this example, the polypeptides of the sequences shown in SEQ ID Nos. 1 to 5 disclosed in the present invention are present in an extract of Mytilus mussel prepared by a specific process, as described in detail below.

[0051] 0.3 g of Mytilus mussel powder was prepared and mixed with 3 ml of double-distilled water (ddH2O) to form a Mytilus mussel aqueous solution (3 ml). After ultrasonic extraction for approximately 1 hour, the mixture was centrifuged (1000 g) and the resulting supernatant was filtered to obtain Mytilus mussel extract.

[0052] 20 μl of the European mussel extract was prepared and subjected to HPLC analysis.

[0053] The analytical conditions for the peptides shown in SEQ ID Nos. 1 to 3 were a C18 column, mobile phases C: acetonitrile + 0.05% TFA (trifluoroacetic acid), D: double-distilled water + 0.05% TFA, and a flow rate of 1.0 ml / min. The mixing ratio of phases C and D is shown in Table 3 below.

[0054] Table 3: HPLC analysis conditions (1) JPEG2026019993000004.jpg41128

[0055] The analytical conditions for the peptides shown in SEQ ID Nos. 4 and 5 were a C18 column, mobile phases C: acetonitrile + 0.05% TFA (trifluoroacetic acid), D: double-distilled water + 0.05% TFA, and a flow rate of 1.0 ml / min. The mixing ratio of phases C and D is shown in Table 4 below.

[0056] Table 4: HPLC analysis conditions (2) JPEG2026019993000005.jpg60128

[0057] Extracts of abalone, cockles, clams, oysters, and corbicula (2 lots) were prepared using the same method as above, and HPLC analysis was also performed after completion.

[0058] Furthermore, the polypeptides of SEQ ID Nos. 1 to 5 disclosed in the present invention were analyzed under the corresponding HPLC analysis conditions described above.

[0059] As shown in Figures 1 and 2, all chromatograms obtained in the above HPLC analysis were compared. The results in Figure 1 indicate that the polypeptides of SEQ ID Nos. 1 to 3 disclosed in the present invention are contained only in the clam extract and the mussel extract. The results in Figure 2 indicate that the polypeptides of SEQ ID Nos. 4 to 5 disclosed in the present invention are contained only in the mussel extract. The retention time of the peptide represented by SEQ ID No. 4 was approximately 8.5 minutes, and the retention time of the peptide represented by SEQ ID No. 5 was approximately 10.9 minutes. These results demonstrate that the polypeptides of SEQ ID Nos. 1 to 5 disclosed in the present invention are unique, do not occur universally in shellfish or fish, and can only be obtained through a specific extraction process.

[0060] Example 4: Analysis of European mussel components

[0061] A Mytilus mussel extract was prepared according to the method described in Example 3. Furthermore, Mytilus mussel was hydrolyzed with alkaline protease to obtain Mytilus mussel hydrolysate.

[0062] The HPLC analysis method was used to determine whether Mytilus mussel raw material (i.e., Mytilus mussel powder), Mytilus mussel extract, and Mytilus mussel hydrolysate each contained the polypeptides of SEQ ID Nos. 1 to 5. The results are shown in Figure 3. The results in Figure 3 demonstrate that Mytilus mussel indeed contains the polypeptides of SEQ ID Nos. 1 to 5 disclosed in the present invention.

[0063] Example 5: Animal Experiments

[0064] Several 10-week-old ICR male mice were prepared and randomly divided into five groups, and the experimental period was four weeks in total.

[0065] Week 1 of the experiment: Mice in each group underwent exercise training three times, including a running test and a grip strength test.

[0066] Weeks 2-4 of the experiment: Feeding was carried out according to the following conditions.

[0067] Group 1: No sample was fed.

[0068] Group 2: Taurine was given at a dose of 300 mg / kg body weight.

[0069] Group 3: The rats were fed with European mussel extract at a dose of 45 mg / kg of body weight.

[0070] Group 4: The rats were fed with European mussel extract at a dose of 90 mg / kg of body weight.

[0071] Group 5: The rats were fed a diet containing 180 mg of European mussel extract per kg of body weight.

[0072] Here, the Mytilus mussel extract administered to the mice in Groups 3 to 5 was prepared according to the method disclosed in Example 3 and contained the polypeptides of SEQ ID Nos. 1 to 5.

[0073] After completing the breeding of each group of mice under the above conditions (confirmation required), the locomotor index measurement was performed. Here, the locomotor index measurement test included the following:

[0074] Mice from each group were allowed to run in a rotating wheel for 15 minutes, and the running distance and speed were measured at 1, 3, and 15 minutes. The results are shown in Figures 4 and 5.

[0075] Mice from each group were hung from a hanging net, pulled vertically downward, and the grip strength of the mice from each group was recorded at the moment they released their forelimbs. The results are shown in Figure 6.

[0076] The cumulative running distance of each group of mice during the test period was recorded, as shown in Figure 7.

[0077] Furthermore, blood samples were collected from the mice in groups 1, 2, and 4 to test the serum testosterone content, and the results are shown in Figure 8.

[0078] The results in Figures 4 to 7 demonstrate that administration of the European mussel extract of the present invention significantly improved the exercise performance of mice compared to Group 1. In terms of explosive power and performance improvement, the performance of Group 5 mice was significantly superior to that of Group 2 mice at 1, 3, and 15 minutes. Meanwhile, although the performance of Groups 3 and 4 mice was inferior to that of Group 2 mice, they were able to effectively maintain their exercise performance and continue to outperform Group 1 mice for extended periods (as shown in Figures 4 and 5). In terms of limb grip strength, the performance of Groups 3 to 5 mice was significantly superior to that of Group 2 mice, with Group 5 mice exhibiting the greatest grip strength (as shown in Figure 6). In terms of daily running distance, the performance of Groups 3 and 4 mice was not significantly different from that of Group 2 mice, while the performance of Group 5 mice was superior to that of Group 2 mice (as shown in Figure 7).

[0079] Furthermore, the results in Figure 8 show that the Mytilus mussel extract disclosed in the present invention effectively improved the serum testosterone content of mice in Group 4, demonstrating that administering the Mytilus mussel extract disclosed in the present invention can achieve the effect of improving athletic performance.

[0080] The above results show that administering the European mussel extract disclosed in the present invention to an individual can effectively improve the individual's athletic performance, regardless of whether the dose is low, medium, or high, and can maintain the individual's athletic performance in an improved state, and that the higher the administered dose, the better the effect of improving the individual's athletic performance.

[0081] The results of Examples 3 and 5 demonstrate that administering any one of the peptides of SEQ ID No. 1 to SEQ ID No. 5 disclosed herein, or a composition containing a peptide of SEQ ID No. 1 to SEQ ID No. 5, such as a Mytilus mussel extract, can improve an individual's athletic performance and muscle mass. This means that the peptides of SEQ ID No. 1 to SEQ ID No. 5 disclosed herein or compositions containing them can be used to treat or prevent sarcopenia or related complications, and can have the effect of improving athletic performance.

[0082] Example 6: Human Experiments

[0083] Each of five subjects received 600 mg of Mytilus mussel extract in the morning. The Mytilus mussel extract was prepared according to the method disclosed in Example 3 and contained polypeptides of SEQ ID Nos. 1-5. After 14 consecutive days of administration of the Mytilus mussel extract, each subject's muscle mass ratio (muscle weight / body weight) and total bimanual grip strength were measured. The results are shown in Figures 9 and 10.

[0084] The results in Figures 9 and 10 show that administration of the Mytilus mussel extract of the present invention for 14 days increased muscle mass by approximately 9.7% and bimanual grip strength by approximately 24%. These results demonstrate that the peptides of SEQ ID Nos. 1 to 5 of the present invention or compositions containing the same, such as Mytilus mussel extract, can indeed improve an individual's athletic performance, muscle content, and muscle strength. Therefore, the peptides of SEQ ID Nos. 1 to 5 of the present invention or compositions containing the same can be used to treat or prevent sarcopenia or related complications, and can improve athletic performance.

Claims

1. A polypeptide having an amino acid sequence that is 95% or more identical to a specific sequence or an amino acid sequence derived therefrom by substituting, deleting, or adding one or more amino acids thereto, wherein the specific sequence is selected from the group consisting of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No.

5.

2. 2. The polypeptide of claim 1, wherein the amino acid sequence is SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No.

5.

3. A use of a polypeptide for preparing a composition for preventing and / or treating sarcopenia or a disease associated therewith, wherein the amino acid sequence of the polypeptide is as described in claim 1.

4. Use of a polypeptide for preparing a composition for preventing and / or treating sarcopenia or a disease associated therewith, wherein the amino acid sequence of the polypeptide is SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No.

5.

5. Use of a group of polypeptides in the preparation of a composition for preventing and / or treating sarcopenia or a disease related thereto, wherein the group of polypeptides includes the polypeptides set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No.

5.

6. Use of a shellfish extract for the preparation of a composition for preventing and / or treating sarcopenia or a disease associated therewith, wherein the shellfish extract contains polypeptides set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No.

5.

7. 7. The use according to claim 6, wherein the shellfish extract is derived from European mussel.

8. 1. Use of a group of polypeptides comprising the polypeptides set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No. 5 in the preparation of a composition for improving athletic performance.

9. 1. Use of a shellfish extract in the preparation of a composition for improving athletic performance, wherein the shellfish extract comprises a polypeptide set forth in SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No.

5.

10. 10. The use according to claim 9, wherein the shellfish extract is derived from European mussel.

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