Polypeptides and use thereof in controlling blood glucose and / or reducing fat

Abstract

To provide polypeptides, and to provide use thereof in controlling blood glucose and / or reducing fat.SOLUTION: The present invention discloses a polypeptide and use thereof for controlling blood glucose and / or reducing fat. The amino acid sequence of the polypeptide includes any of the sequences of SEQ ID No.:1 to SEQ ID No.:4 or a sequence obtained by modifying at least one amino acid therein. The polypeptide disclosed in the present invention has a physiological activity that promotes fat metabolism and blood glucose control, so that by administering an effective amount of the polypeptide disclosed in the present invention or a composition containing the polypeptide to an individual, it is possible to effectively achieve the effect of treating or preventing diseases related to blood glucose imbalance or fat metabolism imbalance.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to polypeptides and their uses, in particular to polypeptides and their uses in controlling blood sugar and / or reducing fat. [Background technology]

[0002] According to a survey, the prevalence of diabetes in Taiwan among people aged 18 and over continues to rise, already exceeding the world average, and there is also a trend of diabetes onset at a younger age. In addition to genetic factors, diabetes is often caused by overeating and high-sugar, high-fat dietary habits. Because dietary habits are difficult to change, many dietary supplements for blood sugar control or weight loss have appeared on the market. Among these, peptides are the main active ingredient in many dietary supplements.

[0003] Peptides are structures composed of amino acids. Peptides are intermediate substances between amino acids and proteins, and their molecular weight is significantly lower than that of proteins, so that they can be absorbed directly or have a high absorption rate after entering the human body. Recent research has found that many peptides have specific physiological functions and activities, such as preventing osteoporosis, suppressing cancer, and anti-aging. Therefore, peptides are now being used more widely in nutritional supplements and skin care products. Currently, most methods for obtaining peptides are by hydrolyzing proteins with enzymes. However, while the enzymatic hydrolysis method can quickly obtain peptides, quality control and improvement of purity are major challenges. 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 controlling blood sugar and / or reducing fat. Specifically, the polypeptide disclosed in the present invention has the ability to activate insulin receptors and stimulate cells to actively transport glucose, thereby enabling blood sugar control and promoting cellular fat oxidation metabolism, and can achieve the effect of effectively controlling blood sugar and / or suppressing fat accumulation.

[0005] A secondary object of the present invention is to provide a polypeptide and its use for controlling blood sugar and / or reducing fat. The polypeptide disclosed in the present invention has the effect of simultaneously controlling blood sugar and promoting fat metabolism, and therefore has the ability to control multiple metabolic indicators, thereby achieving the effect of effectively preventing or treating metabolic syndrome. [Means for solving the problem]

[0006] To achieve the above-mentioned objectives, the amino acid sequence of the polypeptide disclosed in the present invention includes the sequence shown in SEQ ID No.:1, the sequence shown in SEQ ID No.:2, the sequence shown in SEQ ID No.:3, the sequence shown in SEQ ID No.:4, or a sequence obtained by modifying one or more amino acids in any of the above sequences.

[0007] In one embodiment of the present invention, the amino acid sequence of the polypeptide is SEQ ID No.:1.

[0008] In the following examples of the invention, the amino acid sequence of the polypeptide is SEQ ID No.:2.

[0009] In another embodiment of the present invention, the amino acid sequence of the polypeptide is SEQ ID No.:3.

[0010] In yet another embodiment of the present invention, the amino acid sequence of the polypeptide is SEQ ID No.:4.

[0011] The polypeptides disclosed in the present invention can be obtained by artificial synthesis, enzymatic hydrolysis, solvent extraction or other protein preparation techniques well known in the art. For example, the polypeptide having the amino acid sequence of SEQ ID No.:4 can be prepared by artificial synthesis, and can also be obtained by subjecting the polypeptide having the amino acid sequence of SEQ ID No.:3 to an enzymatic hydrolysis process.

[0012] Furthermore, since the polypeptide disclosed in the present invention has the effect of promoting blood glucose control and / or fat metabolism, in one embodiment of the present invention, the polypeptide disclosed in the present invention is used to prepare a composition for controlling blood glucose. That is, when an effective amount of the polypeptide disclosed in the present invention is administered to an individual having symptoms of blood glucose control or an individual already suffering from a blood glucose metabolism imbalance-related disease, the polypeptide disclosed in the present invention can activate insulin receptors and stimulate cells to actively transport glucose, thereby effectively achieving the effects of controlling the individual's blood glucose and delaying the onset of blood glucose-related diseases.

[0013] Here, the blood glucose controlling composition disclosed in the present invention comprises the polypeptide sequence shown in SEQ ID No.:1, the polypeptide shown in SEQ ID No.:2, and the polypeptide shown in SEQ ID No.:3.

[0014] Here, the blood glucose controlling composition disclosed in the present invention comprises the polypeptide sequence shown in SEQ ID No.:1, the polypeptide shown in SEQ ID No.:2, the polypeptide shown in SEQ ID No.:3, and the polypeptide shown in SEQ ID No.:4.

[0015] In another embodiment of the present invention, the polypeptide disclosed in the present invention is used in the preparation of a fat-reducing composition for use in improving the activity of fat metabolism in cells of an individual, thereby suppressing the formation of body fat and fat accumulation in the body even in an individual's high-fat or high-calorie dietary environment, thereby effectively achieving the effects of fat reduction and weight loss.

[0016] The fat reducing compositions disclosed in the present invention include the polypeptide sequence shown in SEQ ID No.:1, the polypeptide shown in SEQ ID No.:2, and the polypeptide shown in SEQ ID No.:3.

[0017] The fat reducing compositions disclosed in the present invention include the polypeptide sequence shown in SEQ ID No.:1, the polypeptide shown in SEQ ID No.:2, the polypeptide shown in SEQ ID No.:3, and the polypeptide shown in SEQ ID No.:4.

[0018] Another embodiment of the present invention discloses a composition, which comprises an effective amount of the above-mentioned polypeptide, wherein the composition can be a medicine, a food, a dietary supplement, etc. By administering the composition to an individual, the effect of treating or preventing a disease caused by blood glucose imbalance or fat metabolism imbalance can be achieved, wherein the disease caused by blood glucose imbalance or fat metabolism imbalance includes hyperglycemia, obesity, diabetes, metabolic syndrome, etc. [Brief description of the drawings]

[0019] [Figure 1] 1 is a liquid chromatogram of a polypeptide extract disclosed in Example 1 of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The amino acid sequence of the polypeptide disclosed in the present invention for use in controlling blood glucose and / or reducing fat includes any one of SEQ ID No.:1 to SEQ ID No.:4 (as shown in Table 1 below), or includes a sequence obtained by modifying one or more amino acids in any one of SEQ ID No.:1 to SEQ ID No.:4. The polypeptide disclosed in the present invention has functions such as activating insulin receptors, promoting active glucose transport in cells, and promoting fat oxidation metabolism in cells. Therefore, when an effective amount of the polypeptide disclosed in the present invention or a composition containing the polypeptide is administered to an individual, it can effectively achieve the effect of reducing fat and / or controlling blood glucose, thereby preventing or treating diseases associated with abnormal blood glucose or fat metabolism, such as metabolic syndrome, hyperglycemia, and diabetes.

[0021] Table 1: Amino acid sequences of the polypeptides disclosed in the present invention JPEG2025063000000002.jpg27151

[0022] In one embodiment of the present invention, the amino acid sequence of the polypeptide disclosed herein for use in blood glucose control and / or fat reduction is SEQ ID No.:1, SEQ ID No.:2, SEQ ID No.:3 or SEQ ID No.:4.

[0023] The term "modification" as used herein refers to substitution, deletion or insertion at least one amino acid position in an amino acid sequence.

[0024] The term "polypeptide" as used herein refers to a low molecular weight protein composed of multiple amino acids.

[0025] The polypeptide disclosed in the present invention can be prepared by techniques for obtaining peptides well known in the art, such as artificial synthesis and enzymatic hydrolysis. The so-called artificial synthesis includes methods such as chemical synthesis and biosynthesis, among which the chemical synthesis method is to obtain a target peptide by connecting multiple amino acids with peptide bonds, and is further divided into liquid phase synthesis, solid phase synthesis, etc. according to the synthesis technique. The biosynthesis method is to produce a target peptide by recombinant microorganisms or recombinant cells, and a nucleic acid molecule capable of expressing the target peptide is transplanted into the recombinant microorganisms or recombinant cells by biotechnology.

[0026] Since the polypeptides disclosed in the present invention are present in animals and plants, they can be isolated from sources such as the peel, pulp, fruit, seeds, leaves, etc. of pumpkin, watermelon, cucumber, loofah, or other Cucurbitaceae plants by isolation techniques well known in the art, such as extraction.

[0027] The polypeptide disclosed in the present invention can be obtained by purification from vegetable or animal proteins, such as pumpkin-derived protein, loofah-derived protein, cucumber-derived protein, or other cucurbit-derived protein, and the commonly used protein purification techniques in the known art include salting out, gel filtration, chromatography, etc. For example, before protein purification, the vegetable or animal protein can be subjected to a process such as enzymatic hydrolysis or extraction, and then the target peptide can be obtained by the above-mentioned protein purification techniques.

[0028] The "composition" disclosed by the present invention refers to a composition containing an effective amount of the polypeptide disclosed by the present invention, such as a dietary supplement, a pharmaceutical, a food, etc., and the effective amount referred to here is 0.01% to 100% of the total amount of the composition. The dosage form of the composition is not limited, and includes, for example, powder, tablet, drop, liquid, etc., and the composition can further include at least one set of ingredients, for example, the composition includes the polypeptide disclosed by the present invention and at least one pharma- ceutically acceptable vehicle. Or, for example, the composition includes the polypeptide disclosed by the present invention and at least one food-acceptable composition ingredient.

[0029] In the following, in order to explain the technical features and effects of the present invention, some test examples will be given together with data.

[0030] Each polypeptide used in the following examples was obtained by artificial synthesis, and was identified by HPLC and LC / MASS to confirm that there were no errors in the sequences. A total of five polypeptides were used in the following examples, and their amino acid sequences are shown in SEQ ID No.:1 to SEQ ID No.:5, respectively, and the molecular weights of each polypeptide are shown in Table 2 below.

[0031] Table 2: Molecular weight of each polypeptide JPEG2025063000000003.jpg32151

[0032] All cells used in the following examples are commercial cell lines and no deposit is required.

[0033] Example 1: Preparation of polypeptide extracts

[0034] Pumpkin seeds were prepared, and after preliminary treatment such as washing and crushing, an extraction step was carried out. Here, the extraction step may be performed by an extraction technique well known to those skilled in the art, such as solvent extraction, supercritical extraction, ultrasonic extraction, etc., but in the operation of this example, the extraction step was performed by supercritical extraction using carbon dioxide as a solvent. Here, the extraction temperature was 30 to 80°C, and the pressure was 8.2 to 50.0 MPa. After the extraction was completed, the crude extract was obtained, and then steps such as disinfection, drying, and powdering were performed to obtain the polypeptide extract disclosed in the present invention. The liquid chromatogram is shown in FIG. 1.

[0035] As can be seen from Figure 1, the polypeptide extract disclosed in the present invention has peaks at all retention times of 8.1 to 8.2, 9.4 to 9.5, 13.0 to 13.1, 14.5 to 14.6, 16.2 to 16.3, 17.9 to 18.0, 19.6 to 19.7, 20.1 to 20.2, 22.0 to 22.1, and 22.6 to 22.7 minutes, indicating that the polypeptide extract disclosed in the present invention is indeed a mixture composed of multiple peptides. Specifically, peptide fraction 1 (P fraction 1) had a retention time of 8.160 min, peptide fraction 2 (P fraction 2) had a retention time of 9.419 min, peptide fraction 3 (P fraction 3) had a retention time of 13.025 min, peptide fraction 4 (P fraction 4) had a retention time of 14.589 min, peptide fraction 5 (P fraction 5) had a retention time of 16.270 min, peptide fraction 6 (P fraction 6) had a retention time of 17.902 min, peptide fraction 7 (P fraction 7) had a retention time of 19.112 min, peptide fraction 8 (P fraction 8) had a retention time of 19.626 min, peptide fraction 9 (P fraction 9) had a retention time of 20.150 min, peptide fraction 10 (P fraction 10) had a retention time of 22.059 min, peptide fraction 11 (P fraction 11) had a retention time of 22.672 min, and peptide fraction 19 (P fraction The retention time of 19) was 23.892 minutes.

[0036] Example 2: Animal testing (1)

[0037] A number of 8-week-old SD rats were prepared and randomly divided into two groups, and the drug STZ (Streptozotocin) was used for intraperitoneal injection (55-75mg / kg) to induce a type 1 diabetes model. One week after induction, blood was collected in a fasting state to test the blood glucose level, and if it exceeded 230mg / dl, the induction was deemed successful and the injection of the drug STZ was immediately stopped. Rats in the blank group were injected with the drug STZ and then fed high-calorie feed. Rats in the polypeptide group were injected with the drug STZ and then fed high-calorie feed and the polypeptide extract disclosed in Example 1 at the same time. Here, the administration dose of the polypeptide was calculated based on the human dose of 300mg / day, i.e., the rat dose was 31.2mg / kg (calculated as 300 / 60*6.25).

[0038] After each group of rats was raised under the above conditions for 8 weeks, the blood glucose level and glycosylated hemoglobin (HbA1c) were examined. The results are shown in Table 3 below.

[0039] As can be seen from the results in Table 3, the blood glucose level of the rats in the polypeptide group was obviously lower than that of the rats in the blank group, and the decrease was about 40%. Furthermore, the glycated hemoglobin of the rats in the polypeptide group was lower than that of the rats in the blank group. This result shows that the polypeptide extract disclosed in the present invention can effectively control the blood glucose level of an individual, and can effectively reduce the possibility of the individual developing diabetes even in the state where the pancreatic islet is damaged.

[0040] Table 3: Blood glucose levels and glycated hemoglobin levels after the test in each group of rats JPEG2025063000000004.jpg16147

[0041] Example 3: Animal testing (2)

[0042] Several 8-week-old SD rats were prepared and randomly divided into two groups, and a low dose of the drug STZ (Streptozotocin) was intraperitoneally injected once every three days to induce and form a type 2 diabetes model. After induction, blood was drawn every day in a fasting state to check the blood glucose level, and when the blood glucose level exceeded 230 mg / dl, the induction was considered successful and the injection of the drug STZ was immediately stopped.

[0043] After starting the injections to the rats in each group, they were simultaneously fed high-calorie feed, and one group of rats (hereinafter referred to as the polypeptide group) was simultaneously administered the polypeptide extract described in Example 1. Here, the administered dose of the polypeptide extract was calculated based on the human dose of 300mg / day, that is, the rat dose was 31.2mg / kg (calculation formula: 300 / 60*6.25). After raising the rats in each group under the above conditions for 8 weeks, the blood glucose level and glycosylated hemoglobin (HbA1c) were respectively examined. The results are shown in Table 4 below.

[0044] As can be seen from the results in Table 4, the blood glucose level of the rats in the polypeptide group was obviously lower than that of the rats in the blank group, with a decrease of about 38.8%. Furthermore, the glycated hemoglobin of the rats in the polypeptide group was lower than that of the rats in the blank group, with a decrease of about 14.3%. This result shows that when an individual suffers from type 2 diabetes or has a high risk of developing type 2 diabetes, the blood glucose level of the individual can be effectively controlled by administering the polypeptide extract disclosed in the present invention, and the effect of preventing or delaying the onset of diabetes or diseases related to blood glucose metabolism imbalance can be achieved.

[0045] Table 4: Blood glucose levels and glycated hemoglobin after the test in each group of rats JPEG2025063000000005.jpg16147

[0046] Example 4: Animal testing (3)

[0047] A number of 8-week-old SD rats were prepared and randomly divided into two groups, the rats in the blank group were given only high-calorie feed, and the rats in the polypeptide group were given high-calorie feed and the polypeptide extract described in Example 1 at the same time. Here, the administration dose of the polypeptide extract was calculated based on the human dose of 300 mg / day, that is, the rat dose was 31.2 mg / kg (calculated as 300 / 60*6.25).

[0048] After each group of rats was raised under the above conditions for 8 weeks, the rats were weighed and sacrificed to measure the weight of abdominal fat. The results are shown in Table 5 below.

[0049] As can be seen from the results in Table 5, both the body weight and abdominal fat weight of the polypeptide group rats were significantly lower than those of the blank group rats; specifically, the body weight and abdominal fat weight of the polypeptide group rats were 17.9% and 27.7%, respectively, lower than those of the blank group rats.

[0050] This result indicates that when an individual continues to eat a high-fat or high-calorie diet and at the same time the polypeptide extract disclosed in the present invention is administered, it can not only suppress fat accumulation but also increase fat metabolism, thereby achieving the effect of reducing weight and body fat, or preventing a significant increase in body weight. In other words, the polypeptide extract disclosed in the present invention can be used to prevent and / or treat fat metabolism-related diseases such as obesity and metabolic syndrome.

[0051] Table 5: Body weight and abdominal fat of rats in each group after the test JPEG2025063000000006.jpg16147

[0052] Example 5: Cellular testing

[0053] As can be seen from the results of the above examples, the polypeptide extract disclosed in Example 1 has activities such as lowering glycated hemoglobin, controlling blood sugar, and increasing fat metabolism. Therefore, the polypeptides shown in SEQ ID No.:1 to SEQ ID No.:5 were isolated therefrom, their sequences were determined, and they were prepared by artificial synthesis, and the following cell test was carried out.

[0054] Mouse myoblast cell line C2C12 was prepared, divided into groups, and cultured for 24 hours. Human insulin (MedChemExpress), polypeptide 1 disclosed in the present invention (SEQ ID No.: 1), polypeptide 2 disclosed in the present invention (SEQ ID No.: 2), polypeptide 3 disclosed in the present invention (SEQ ID No.: 3), polypeptide 4 disclosed in the present invention (SEQ ID No.: 4), and polypeptide 5 (amino acid sequence code SEQ ID No.: 5) as a control were added to the medium to a final concentration of 20 μL / mL, and each cell group was cultured for another 24 hours. The medium of each cell group was removed, and the cells were washed with phosphate buffer, and then RIPA buffer containing phosphatase inhibitor was added. The cells were then left to stand in a refrigerator for 30 minutes to react, and then thawed. The cells were then subjected to 5 minutes of shaking extraction with an ultrasonic oscillator, and centrifuged to obtain the supernatant of the nuclear cell group. The results were analyzed by Western blotting to obtain the changes in the expression levels of p-IR / IR, p-Akt, GLUT4, mTOR, PPARα, and p-AMPK in each cell group. The results are shown in Table 6 below.

[0055] Table 6: Detection results of changes in expression of proteins related to blood glucose control, insulin receptor activation, and fat oxidation metabolism in each cell group. JPEG2025063000000007.jpg77151

[0056] As can be seen from the results in Table 6, polypeptide 1 having the amino acid sequence SEQ ID No.:1, polypeptide 2 having the amino acid sequence SEQ ID No.:2, and polypeptide 3 having the amino acid sequence SEQ ID No.:3 disclosed in the present invention were able to promote insulin receptor phosphorylation, increase the expression levels of phosphorylated AKT and GLUT4, and improve the expression levels of PGC-1α and PPARα, which are important proteins in the fat oxidation pathway, without insulin induction. On the other hand, polypeptide 4 having the amino acid sequence SEQ ID No.:4 disclosed in the present invention did not show a significant increase in the expression level of phosphorylated AKT, but was also able to promote insulin receptor phosphorylation, increase the expression level of GLUT4, and improve the expression levels of PGC-1α and PPARα, which are important proteins in the fat oxidation pathway, without insulin induction. Polypeptide 5 having the amino acid sequence of SEQ ID No.:5 was unable to improve the expression level of phosphorylated AKT, and was less effective in improving the expression levels of GLUT4, PGC-1α and PPARα, compared with each of the polypeptides having the amino acid sequences of SEQ ID No.:1 to SEQ ID No.:4. As can be seen from the above results, each of the polypeptides having the amino acid sequences of SEQ ID No.:1 to SEQ ID No.:4 disclosed in the present invention was superior to insulin in the expression levels of p-IR / IR, p-Akt, GLUT4, mTOR, PPARα and p-AMPK.

[0057] As can be seen from the results in Table 6, each of the polypeptides having amino acid sequences of SEQ ID No.:1 to SEQ ID No.:4 disclosed in the present invention can activate insulin receptors, promote active glucose transport in cells to control blood glucose, and promote fat oxidation metabolism in cells, and can be used to control blood glucose and reduce body weight in individuals. In other words, by administering an effective amount of each of the polypeptides having amino acid sequences of SEQ ID No.:1 to SEQ ID No.:4 disclosed in the present invention or a composition containing any of the above-mentioned polypeptides to an individual, it is possible to treat and / or prevent diseases related to blood glucose imbalance or abnormal fat metabolism, such as diabetes, hyperglycemia, obesity, and metabolic syndrome.

Claims

1. The amino acid sequence is a polypeptide comprising a sequence represented by SEQ ID No.: 1, a sequence represented by SEQ ID No.: 2, a sequence represented by SEQ ID No.: 3, a sequence represented by SEQ ID No.: 4, or a sequence obtained by modifying one or more amino acids in any of the above sequences.

2. The polypeptide of claim 1, wherein the amino acid sequence is selected from the group consisting of the sequence shown in SEQ ID No.: 1, the sequence shown in SEQ ID No.: 2, the sequence shown in SEQ ID No.: 3, and the sequence shown in SEQ ID No.:

4.

3. A composition comprising an effective amount of a polypeptide, the amino acid sequence of the polypeptide being selected from the group consisting of one or more of the following sequences: SEQ ID No.: 1, SEQ ID No.: 2, SEQ ID No.: 3, and SEQ ID No.:

4.

4. The composition of claim 3, characterized in that the composition comprises the polypeptides shown in SEQ ID No.: 1, SEQ ID No.: 2, SEQ ID No.: 3, and SEQ ID No.:

4.

5. A composition for controlling blood sugar comprising the polypeptide of claim 2.

6. 6. The composition of claim 5, wherein the composition comprises a polypeptide as set forth in SEQ ID No.:1, SEQ ID No.:2, SEQ ID No.:3, and SEQ ID No.:

4.

7. A composition for reducing fat comprising the polypeptide of claim 2.

8. 8. The composition of claim 7, wherein the composition comprises a polypeptide as set forth in SEQ ID No.: 1, SEQ ID No.: 2, SEQ ID No.: 3, and SEQ ID No.: 4.

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