Polypeptides and their applications in promoting muscle growth and improving athletic 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 low molecular weight proteins and their uses, and particularly to polypeptides and their uses for promoting muscle growth and improving exercise performance.

Background Art

[0002] So-called "sarcopenia" refers to a disease in which the amount and function of skeletal muscle throughout the body continue to decline. Patients suffering from sarcopenia are unable to stand up or walk for a long time, lift heavy objects, and are prone to falling due to the decrease in muscle mass, which affects their life and behavior. In severe cases, they require nursing care, cannot live alone, and the risk of death increases. Sarcopenia is more likely to occur in the elderly, but even the young can suffer from it. The main causes are insufficient protein intake, malnutrition, lack of exercise habits, or inappropriate diets.

[0003] Currently, there are still no clinically available drugs that can treat sarcopenia. Clinicians can only recommend nutritional supplements to patients and increase muscle strength through exercise. However, many patients avoid exercise because they have no muscle strength or poor exercise performance. There are many nutritional foods containing a lot of protein on the market, which claim to be able to prevent sarcopenia. However, supplementing protein cannot improve muscle mass and exercise performance, but only delay the decrease in muscle mass, and the effect of effectively treating or preventing sarcopenia or its complications cannot be achieved.

Summary of the Invention

Problems 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 exercise performance, which can effectively achieve strengthening muscle mass and promoting muscle growth by daily supplementation of the polypeptide disclosed by the present invention, and achieve the effect of preventing or treating diseases caused by muscle deficiency or muscle damage.

[0005] Another objective of the present invention is to provide polypeptides and their applications for promoting muscle growth and improving athletic performance, that is, by administering the polypeptides or compositions thereof disclosed in the present invention to an individual, the individual's explosive power and endurance in exercise will be improved. [Means for solving the problem]

[0006] To achieve the above objectives, the present invention discloses a polypeptide and its uses, wherein the amino acid sequence of the polypeptide includes an amino acid sequence having 95% or more identical equivalentity to a specific sequence or an amino acid sequence derived by substituting, deleting, or adding one or more amino acids thereof, of which 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 composed of one of the sequences SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, or SEQ ID No. 5. [Effects of the Invention]

[0008] Since the polypeptides disclosed in this invention have activity that promotes muscle synthesis and proliferation, administering an effective amount of the polypeptides disclosed in this invention or a composition containing the polypeptides to an individual can effectively improve or enhance the individual's muscle mass and strength.

[0009] Specifically, when an individual to be administered the polypeptide or a composition containing the polypeptide disclosed in the present invention exhibits symptoms of muscle deficiency, administering an effective amount to the individual can achieve the effect of treating and / or preventing diseases associated with muscle deficiency. Here, diseases associated with muscle deficiency include, for example, sarcopenia, fractures, and osteoporosis.

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

[0011] Furthermore, the polypeptides disclosed in this invention may be derived from shellfish extracts, such as extracts from the European mussel (Mytilus edulis) or the clam (Mytilus edulis). [Brief explanation of the drawing]

[0012] [Figure 1] This shows a comparison of chromatograms after HPLC analysis of different seafood extracts and polypeptides shown as SEQ ID No. 1-3 disclosed in this invention. [Figure 2] This shows a comparison of chromatograms after HPLC analysis of different seafood extracts and polypeptides shown in SEQ ID No. 4-5 disclosed in this invention. [Figure 3] This is the result of comparing the HPLC chromatograms of European mussel raw material, European mussel extract, and European mussel hydrolysate. [Figure 4] This is the total distance traveled by mice in each group, measured at 1 minute, 3 minutes, and 15 minutes. [Figure 5] These are the running speeds of the mice in each group, analyzed at 1 minute, 3 minutes, and 15 minutes. [Figure 6] This represents the limb grip strength of the mice in each group. [Figure 7] This represents the total distance run by each group of mice each day during the test period. [Figure 8] This represents the testosterone content in the serum of mice from each group. [Figure 9] This is an analysis of the change in muscle mass for each subject during the test period (14 days). [Figure 10] This is the sum of the grip strength tests performed on each subject's hands. [Modes for carrying out the invention]

[0013] This invention discloses polypeptides and their uses in promoting muscle growth and improving athletic performance. The polypeptides disclosed are low-molecular-weight molecules composed of 9 to 12 amino acids and have the activity to control the expression of proteins such as p-AMPK, AKT, and mTOR in the muscle growth pathway. Therefore, by administering an effective amount of the polypeptides disclosed or compositions containing the polypeptides to an individual, it is possible to promote muscle growth and muscle strength in the individual, thereby achieving the effects of preventing or treating diseases related to muscle damage and improving athletic performance.

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

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

[0016] In another embodiment of the present invention, the polypeptide sequence is a sequence that is 95% or more identical to any of the sequences shown 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 one or more amino acids in any of the sequences shown in SEQ ID No. 1 to SEQ ID No. 5.

[0017] In one embodiment of the present invention, the polypeptide may be prepared by an artificial synthesis method well known in the art, or it may be produced on a bioproduction platform. Specifically, a nucleic acid molecule that can be transcribed into the polypeptide by a biotechnology well known in the art can be introduced into a recombinant vector, the recombinant vector can then be introduced into a host cell and expressed to obtain a product containing the polypeptide, and then the polypeptide can be obtained by purification and separation technology.

[0018] In another embodiment of the present invention, the polypeptide disclosed by the present invention may be derived from a shellfish extract, such as an extract of Mytilus edulis. The shellfish extract is obtained by performing an extraction step on a specific shellfish with a predetermined extraction solvent. Here, the extraction step is preferably a solvent extraction method, an ultrasonic extraction method, a supercritical extraction method, or a combination of at least two of the above methods. Furthermore, the extraction solvent is water, ethanol, methanol, or other solutions well-known in the technical field to which the present invention belongs. [[ID=^{4}]]

[0019] In another embodiment of the present invention, the polypeptide disclosed by the present invention may be derived from a shellfish hydrolyzate, such as a hydrolyzate of Mytilus edulis. Here, the shellfish hydrolyzate is obtained by performing a hydrolysis step with a hydrolyzing enzyme, and the hydrolyzing enzyme is preferably an alkaline protease or other hydrolyzing enzymes well-known in the art and acceptable in the food industry.

[0020] In one embodiment of the present invention, the composition contains a polypeptide represented by 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 contains polypeptides represented by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5.

[0022] The term "Mytilus edulis" is a kind of shellfish seafood also called "Mussel" or "mussel".

[0023] The term "Mytilus edulis powder" is obtained through the steps of boiling Mytilus edulis sufficiently, removing the shells, drying, and pulverizing. [[ID=^{23}]]

[0024] The term "sarcopenia" refers to a disease characterized by a continuous decline in the mass and function of skeletal muscle throughout the body. In clinical practice, sarcopenia is assessed based on factors such as an individual's ability to lift heavy objects, whether or not they require assistance while walking, the difficulty of standing up from a chair, the number of steps they can climb on stairs, and the number of falls.

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

[0026] The term "effective dose" refers to the amount of intake required to prevent, improve, or treat the disease or symptom 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, it is possible to achieve improvement of the disease or symptoms being addressed, or to delay the worsening of the disease being addressed.

[0028] The term "prevention" means that when an individual is at risk of contracting a disease that affects them, administering the polypeptide disclosed in this invention or a composition containing the polypeptide to the individual can prevent the onset of the disease or delay the timing of the onset of the disease that affects them.

[0029] The term "composition" refers to a composition whose main active ingredient is a polypeptide or a substance containing a polypeptide disclosed in the present invention. Such compositions can be manufactured in various product forms and manners according to demand, such as nutritional supplements, foods, pharmaceuticals, and beverages. Furthermore, they can be prepared in various dosage forms, such as tablets, powder packets, and jellies, depending on factors such as their product form, target population, and market sales.

[0030] The technical features and effects of the present invention will be explained below with reference to several experimental examples.

[0031] Since the cells used in the following examples are readily available to those skilled in the art, patent deposit is not required.

[0032] The animal experiments conducted in the following examples comply with ethical guidelines for animal experimentation.

[0033] The operating conditions, data, dosages, and other figures 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 general knowledge or experimental practice, that the data or figures disclosed in the following examples include an acceptable margin of error. That is, according to common practice in the art to which the present invention belongs, the acceptable margin of error is within ±10% of the disclosed 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 illustrative only. That is, according to general knowledge of the present invention, the dosages shown in the following examples can be converted depending on the type of substance administered and the conditions under which it is administered, and the scope of protection of the present invention is not limited thereto.

[0035] Example 1: Preparation of synthetic sequence

[0036] The polypeptides indicated by sequences SEQ ID No. 1 to SEQ ID No. 5 were synthesized using an artificial method, and their sequences were confirmed to be free of errors. The results are shown in Table 1.

[0037] Table 1: Information on each polypeptide JPEG0007905130000001.jpg47150

[0038] Example 2: Cell test

[0039] C2C12 myoblast cells 1 x 10 4Seeds were seeded in 24-well plates at a concentration of counts / mL and cultured at 37°C under 5% CO2 conditions using the culture medium described below, with the culture medium changed every 2-3 days.

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

[0041] After culturing C2C12 myoblasts to 90% density, the culture medium was replaced with DMEM medium containing 2% horse serum. The culture medium was changed once every 48 hours. After a total of 4 days of culture, if differentiation and formation of myotubes were observed in the C2C12 myoblasts, the subsequent group classification test could be performed.

[0042] C2C12 myoblasts were cultured in groups based on the following conditions.

[0043] The first group is the blank group.

[0044] Group 2 was the positive control group, and they received 100 nM of insulin.

[0045] The third group consisted of samples, to which 400 μg / ml of the sample was added. Here, the sample is the polypeptide of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 from Example 1.

[0046] After culturing the cells from each of the above groups for 24 hours, the cells were collected, proteins were extracted, and the expression levels of the following proteins: AMPK, p-AMPK, mTOR, p-mTOR, AKT, and p-AKT were analyzed by Western blotting. The relative expression ratios compared to the cell group without any added sample (blank group) were then calculated. The results are shown in Table 2 below. Of these, the values ​​for p-AMPK / AMPK, P-AKT / AKT, and P-mTOR / mTOR in the blank group are all 1.

[0047] Table 2: Protein analysis results for each polypeptide JPEG0007905130000002.jpg53149

[0048] As can be seen from the results in Table 2, each polypeptide disclosed in the present invention can have the activity to enhance 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 major factors that control muscle proliferation in the muscle proliferation mechanism. Therefore, the above results demonstrate that any polypeptide of the sequences shown in SEQ ID No. 1 to 5 disclosed in the present invention has the activity to improve the expression of p-AMPK, AKT, and mTOR. It has been shown that when any polypeptide of the sequences shown in SEQ ID No. 1 to 5 disclosed in the present invention is administered to an individual in an effective amount, it is possible to achieve the effect of preventing or treating sarcopenia or related symptoms, or improving athletic performance, by promoting muscle cell regeneration, breakdown of damaged muscle, and promotion of muscle cell proliferation.

[0049] Example 3: Preparation of seafood extracts

[0050] In this embodiment, polypeptides with sequences indicated by SEQ ID No. 1 to 5 disclosed in the present invention appear in an extract of European mussel prepared by a predetermined process. Details are described below.

[0051] 0.3 g of European mussel powder was prepared and mixed with 3 ml of redistilled water (ddH2O) to form a European mussel aqueous solution (3 ml). Ultrasonographic extraction was performed for about 1 hour, and the supernatant obtained by centrifugation (1000 g) was filtered to obtain a European mussel extract.

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

[0053] Here, the analytical conditions for the peptides shown in SEQ ID No. 1-3 were as follows: C18 column, mobile phase C was acetonitrile + 0.05% TFA (Trifluoroacetic acid), and mobile phase D was redistilled water + 0.05% TFA, with a flow rate of 1.0 ml / min. The mixing ratio of phase C and phase D is as shown in Table 3 below.

[0054] Table 3: HPLC analysis conditions (1) JPEG0007905130000003.jpg41128

[0055] Here, the analytical conditions for the peptides shown in SEQ ID No. 4-5 were as follows: C18 column, mobile phase C was acetonitrile + 0.05% TFA (Trifluoroacetic acid), and mobile phase D was redistilled water + 0.05% TFA, with a flow rate of 1.0 ml / min. The mixing ratio of phase C and phase D is as shown in Table 4 below.

[0056] Table 4: HPLC analysis conditions (2) JPEG0007905130000004.jpg60128

[0057] Using the same method as described above, extracts of abalone, ark clams, cockles, oysters, and freshwater clams (2 lots) were prepared, and HPLC analysis was also performed after completion.

[0058] Furthermore, the polypeptides with SEQ IDs 1 to 5 disclosed in this invention were analyzed under the corresponding HPLC analytical conditions described above.

[0059] As shown in Figures 1 and 2, all chromatograms obtained by the above HPLC analysis were compared. From the results in Figure 1, it can be seen that polypeptides with SEQ IDs 1-3 disclosed in this invention are contained only in the clam extract and the mussel extract. From the results in Figure 2, it can be seen that polypeptides with SEQ IDs 4-5 disclosed in this invention are contained only in the mussel extract. Here, the retention time of the peptide shown in SEQ ID 4 was approximately 8.5 minutes, and the retention time of the peptide shown in SEQ ID 5 was approximately 10.9 minutes. From the above results, it was shown that polypeptides with SEQ IDs 1-5 disclosed in this invention are unique, do not appear universally in shellfish or fish, and can only be obtained through a specific extraction process.

[0060] Example 4: Component analysis of European mussels

[0061] A mussel extract was prepared according to the method shown in Example 3. Furthermore, the mussels were hydrolyzed with an alkaline protease to obtain a mussel hydrolysate.

[0062] The presence or absence of polypeptides with SEQ IDs 1-5 in each of the following materials was determined by HPLC analysis: European mussel raw material (i.e., European mussel powder), European mussel extract, and European mussel hydrolysate. The results are shown in Figure 3. The results in Figure 3 demonstrate that the European mussel indeed contains polypeptides with SEQ IDs 1-5 disclosed in this invention.

[0063] Example 5: Animal experiment

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

[0065] Week 1 of the experiment: Each group of mice underwent three sessions of exercise training. The exercise training included 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 feeding of the samples.

[0068] Group 2: Taurine was administered. The dose was 300 mg / kg-body weight.

[0069] Group 3: These groups were fed European mussel extract. The dosage was 45 mg / kg-body weight.

[0070] Group 4: The group was fed European mussel extract. The dose was 90 mg / kg-body weight.

[0071] Group 5: The group was fed European mussel extract. The dose was 180 mg / kg-body weight.

[0072] Here, the mussel extract administered to groups 3-5 of mice was prepared according to the method disclosed in Example 3 and contains polypeptides with SEQ ID No. 1-5.

[0073] After completing the rearing of the mice in each group under the above conditions (to be confirmed), motor indicator measurements were performed. The motor indicator measurement tests included the following:

[0074] The mice in each group were made to run in a wheel for 15 minutes, and the distance and speed covered were measured at 1 minute, 3 minutes, and 15 minutes, respectively. The results are shown in Figures 4 and 5.

[0075] Each group of mice was suspended from a net, and the mice were pulled vertically downwards. The grip strength at the moment each mouse released its forelimbs was recorded. The results are shown in Figure 6.

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

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

[0078] The results in Figures 4 to 7 show that administration of the European mussel extract disclosed in this invention significantly improves the motor performance of mice compared to the first group of mice. In terms of explosive power and improvement in motor performance, the performance of the fifth group of mice was clearly superior to that of the second group of mice at 1 minute, 3 minutes, and 15 minutes. On the other hand, although the performance of the third and fourth groups of mice did not reach that of the second group of mice, the mice's motor performance was effectively maintained and continued to outperform the first group of mice for a long period of time (as shown in Figures 4 and 5). In terms of limb grip strength, the performance of the third to fifth groups of mice was clearly superior to that of the second group of mice, with the fifth group of mice showing the best grip strength (as shown in Figure 6). In terms of daily running distance, the performance of the third and fourth groups of mice was not significantly different from that of the second group of mice, while the performance of the fifth group of mice surpassed that of the second group of mice (as shown in Figure 7).

[0079] Furthermore, the results shown in Figure 8 indicate that the mussel extract disclosed in this invention effectively increased the serum testosterone content of the mice in group 4, demonstrating that administering the mussel extract disclosed in this invention can improve athletic performance.

[0080] The results above demonstrate that administering the European mussel extract disclosed in this invention to an individual effectively enhances the individual's athletic performance, regardless of whether the dose is low, medium, or high, and can maintain the improved athletic performance. Furthermore, it was shown that the higher the dose administered, the better the effect on improving the individual's athletic performance.

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

[0082] Example 6: Human experiment

[0083] Each of the five subjects was administered 600 mg of European mussel extract in the morning. The European mussel extract was prepared according to the method disclosed in Example 3 and contained polypeptides with SEQ ID No. 1 to 5. After administering the European mussel extract for 14 consecutive days, the percentage of muscle mass (muscle weight / body weight) and the sum of the grip strength of both hands were measured for each subject. The results are shown in Figures 9 and 10.

[0084] The results shown in Figures 9 and 10 indicate that 14 days of administration of the European mussel extract disclosed in this invention resulted in an increase of approximately 9.7% in muscle mass and an increase of approximately 24% in bilateral grip strength. These results demonstrate that the peptides of SEQ ID No. 1 to SEQ ID No. 5 disclosed in this invention, or compositions containing them, such as European mussel extract, can indeed improve an individual's athletic performance, muscle content, and muscle strength. Therefore, the peptides of SEQ ID No. 1 to SEQ ID No. 5 disclosed in this invention, or compositions containing them, can be used to treat or prevent sarcopenia or its related complications, and can be used to improve athletic performance.

Claims

1. A composition for preventing and / or treating sarcopenia or related diseases, comprising a shellfish extract derived from the European mussel, comprising the polypeptides represented by SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 and SEQ ID No.

5.

2. A composition for improving athletic performance, comprising a shellfish extract derived from the European mussel, comprising polypeptides represented by SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, and SEQ ID No. 5.

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