Polypeptides and their applications for controlling blood sugar 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, particularly to polypeptides and their use for controlling blood glucose and / or reducing fat.

Background Art

[0002] According to investigations, the prevalence of diabetes among nationals aged 18 and above in Taiwan has been continuously increasing, already exceeding the world average diabetes prevalence, and a trend of diabetes becoming younger is also observed. Among the causes of diabetes, in addition to genetics, it is often caused by refined, high-sugar, and high-fat eating habits. Since it is difficult to change eating habits, many nutritional supplements related to blood glucose control or weight loss have emerged in the market. Among them, peptides are the main active ingredients of many nutritional supplements.

[0003] A peptide is a structure composed of amino acids. A peptide is an intermediate substance between amino acids and proteins, and its molecular weight is significantly lower than that of proteins, so it can be absorbed as it is or has an excellent absorption rate after entering the human body. In recent research, it has been discovered that many peptides have specific physiological functions and activities respectively. For example, prevention of osteoporosis, suppression of cancer, anti-aging, etc. Therefore, currently peptides are being used more widely in nutritional supplements and skin care products. Currently, most methods for obtaining peptides adopt the method of hydrolyzing proteins with enzymes. However, while the enzymatic hydrolysis method can quickly obtain peptides, quality control and purity improvement become major issues.

Summary of the Invention

Problems to be Solved by the Invention

[0004] The main object of the present invention is to provide polypeptides and applications for using them to control blood glucose and / or reduce fat. Specifically, the polypeptides disclosed in the present invention have the ability to activate insulin receptors and stimulate cells to actively transport glucose, thereby enabling blood glucose control and promotion of cellular fatty acid metabolism, and thus achieving the effect of effectively suppressing blood glucose control and / or fat accumulation.

[0005] A secondary object of the present invention is to provide polypeptides and uses for controlling blood glucose and / or reducing fat. Since the polypeptides disclosed by the present invention have the effect of simultaneously controlling blood glucose and promoting fat metabolism, the polypeptides disclosed by the present invention have the ability to control multiple metabolic indicators and can achieve the effect of effectively preventing or treating metabolic syndrome. [Means for solving the problem]

[0006] To achieve the above objectives, the amino acid sequences of the polypeptides disclosed in the present invention include the sequence indicated by SEQ ID No.:1, the sequence indicated by SEQ ID No.:2, the sequence indicated by SEQ ID No.:3, the sequence indicated by SEQ ID No.:4, or sequences 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 embodiment of the present 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 this invention can be obtained by artificial synthesis, enzymatic hydrolysis, solvent extraction, or other protein preparation techniques well known in the art. For example, a polypeptide with an amino acid sequence of SEQ ID No.:4 can be prepared by artificial synthesis, and a polypeptide with an amino acid sequence of SEQ ID No.:3 can also be obtained by an enzymatic hydrolysis process.

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

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

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

[0015] In another embodiment of the present invention, the polypeptide disclosed by the present invention is used to prepare a fat-reducing composition used to improve the activity of fat metabolism in individual cells, 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, and effectively achieving fat reduction and weight loss effects.

[0016] The fat-reducing composition disclosed in this invention comprises a polypeptide sequence indicated by SEQ ID No.:1, a polypeptide indicated by SEQ ID No.:2, and a polypeptide indicated by SEQ ID No.:3.

[0017] The fat-reducing composition disclosed in this invention comprises a polypeptide sequence indicated by SEQ ID No.:1, a polypeptide indicated by SEQ ID No.:2, a polypeptide indicated by SEQ ID No.:3, and a polypeptide indicated by SEQ ID No.:4.

[0018] Another embodiment of the present invention discloses a composition containing an effective amount of the polypeptide described above, which may be a pharmaceutical, food, or nutritional supplement. By administering the composition to an individual, it is possible to achieve the effect of treating or preventing diseases caused by blood glucose imbalance or lipid metabolism imbalance, which include hyperglycemia, obesity, diabetes mellitus, metabolic syndrome, and the like. [Brief explanation of the drawing]

[0019] [Figure 1] This is a liquid chromatogram of a polypeptide extract disclosed in Example 1 of the present invention. [Modes for carrying out the invention]

[0020] The amino acid sequences of the polypeptides used for controlling blood glucose and / or reducing fat disclosed by the present invention include any one of the sequences of SEQ ID No.: 1 to SEQ ID No.: 4 (as shown in Table 1 below), or include sequences obtained by modifying one or more amino acids possessed by any one of the sequences of SEQ ID No.: 1 to SEQ ID No.: 4. Since the polypeptides disclosed by the present invention have functions such as activating insulin receptors, promoting cells to actively transport glucose, and promoting the fatty acid metabolism of cells, when an effective amount of the polypeptides disclosed by the present invention or a composition containing the polypeptides is administered to an individual, the effects of reducing fat and / or controlling blood glucose can be effectively achieved, thereby preventing or treating blood glucose or fat metabolism disorder-related diseases such as metabolic syndrome, hyperglycemia, diabetes, etc.

[0021] Table 1: Amino acid sequences of the polypeptides disclosed by the present invention JPEG0007880154000001.jpg27151

[0022] In one embodiment of the present invention, the amino acid sequences of the polypeptides used for controlling blood glucose and / or reducing fat disclosed by the present invention are SEQ ID No.: 1, SEQ ID No.: 2, SEQ ID No.: 3 or SEQ ID No.: 4.

[0023] The "modification" referred to by the present invention means performing substitution, deletion or insertion at at least one amino acid position in the amino acid sequence.

[0024] The "polypeptide" referred to by the present invention means a low molecular weight protein composed of a plurality of amino acids.

[0025] The polypeptides disclosed by the present invention can be prepared by techniques for obtaining peptides well-known in the art, such as chemical synthesis methods and enzymatic hydrolysis methods. So-called chemical synthesis methods include methods such as chemical synthesis and biosynthesis. Among them, the chemical synthesis method connects a plurality of amino acids by peptide bonds to obtain the target peptide, and can be further divided into liquid-phase synthesis, solid-phase synthesis, etc. according to the synthesis technology. The biosynthesis method produces the target peptide by recombinant microorganisms or recombinant cells. In the recombinant microorganisms or recombinant cells, a nucleic acid molecule capable of expressing the target peptide has been transplanted into the microorganisms or cells by biotechnology.

[0026] Since the polypeptides disclosed by the present invention are present in animals and plants, they can be separated from raw materials such as the peel, pulp, fruit, seeds, leaves, etc. of pumpkins, watermelons, cucumbers, loofahs or other cucurbitaceous plants by separation techniques well-known in the art such as extraction.

[0027] The polypeptides disclosed by the present invention can be obtained by purification from plant proteins or animal proteins, such as pumpkin-derived proteins, loofah-derived proteins, cucumber-derived proteins or proteins derived from other cucurbitaceous plants. Protein purification techniques commonly used in known technologies include salting-out methods, gel filtration methods, chromatography methods, etc. For example, before performing protein purification, processes such as enzymatic hydrolysis and extraction can be performed on the plant protein or animal protein first, and then the target peptide can be obtained by the above-mentioned protein purification techniques.

[0028] The “composition” disclosed in this invention refers to a substance containing an effective amount of the polypeptide disclosed in this invention, such as a nutritional supplement, a pharmaceutical product, or a food product, where the effective amount is 0.01% to 100% of the total amount of the composition. The dosage form of the composition is not limited, but includes, for example, powders, tablets, drops, liquids, etc., and the composition may further contain at least one set of components, for example, the composition containing the polypeptide disclosed in this invention and at least one pharmaceutically acceptable vehicle. Alternatively, for example, the composition may contain the polypeptide disclosed in this invention and at least one food-acceptable component.

[0029] The technical features and effects of the present invention will be explained below by providing several test examples along with data.

[0030] Each polypeptide used in the following examples was obtained through artificial synthesis, and its sequence was confirmed to be correct by HPLC and LC / MASS. A total of five polypeptides were used in the following examples, and their amino acid sequences are indicated by SEQ ID No.:1 to SEQ ID No.:5, respectively. The molecular weight of each polypeptide is shown in Table 2 below.

[0031] Table 2: Molecular weight of each polypeptide JPEG0007880154000002.jpg32151

[0032] The cells used in the following examples are all commercial cell lines and do not require deposit.

[0033] Example 1: Preparation of polypeptide extracts

[0034] Pumpkin seeds were prepared and subjected to preliminary processing steps such as washing and grinding, followed by an extraction process. While the extraction process could be carried out using extraction techniques well known to those skilled in the art, such as solvent extraction, supercritical fluid extraction, or ultrasonic extraction, in this example, the extraction was performed using supercritical fluid extraction with carbon dioxide as the solvent. The extraction temperature was 30-80°C, and the pressure was 8.2-50.0 MPa. After extraction, the crude product was obtained, followed by disinfection, drying, and powdering processes to obtain the polypeptide extract disclosed in this invention. The liquid chromatogram is shown in Figure 1.

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

[0036] Example 2: Animal Testing (1)

[0037] Several 8-week-old SD rats were prepared and randomly divided into two groups. Type 1 diabetes models were induced by intraperitoneal injection (55-75 mg / kg) of the drug STZ (Streptozotocin). Blood glucose levels were measured by fasting for one week after induction. If the blood glucose level exceeded 230 mg / dl, induction was considered successful, and STZ injection was immediately stopped. The blank group rats were given a high-calorie diet after being injected with STZ. The polypeptide group rats were given a high-calorie diet and the polypeptide extract disclosed in Example 1 simultaneously after being injected with STZ. Here, the polypeptide dose was calculated by converting it to the human dose of 300 mg / day, i.e., the rat dose was 31.2 mg / kg (calculation formula: 300 / 60 * 6.25).

[0038] After raising rats from each group under the conditions described above for 8 weeks, blood glucose levels and glycated hemoglobin (HbA1c) were examined, respectively. The results are shown in Table 3 below.

[0039] As can be seen from the results in Table 3, the blood glucose levels of the polypeptide group rats were clearly lower than those of the blank group rats, with a decrease of approximately 40%. Furthermore, the glycated hemoglobin levels of the polypeptide group rats were lower than those of the blank group rats. These results demonstrate that the polypeptide extract disclosed in this invention can effectively control the blood glucose levels of individuals and effectively reduce the likelihood of developing diabetes even in individuals with damaged pancreatic islets.

[0040] Table 3: Blood glucose levels and glycated hemoglobin levels in each rat group after the experiment. JPEG0007880154000003.jpg16147

[0041] Example 3: Animal Testing (2)

[0042] Several 8-week-old SD rats were prepared and randomly divided into two groups. A type 2 diabetes model was induced and formed by administering low doses of the drug STZ (Streptozotocin) intraperitoneally every three days. Blood glucose levels were measured daily in a fasting state after induction. When blood glucose levels exceeded 230 mg / dl, induction was considered successful, and STZ injections were immediately stopped.

[0043] After initiating injections into each group of rats, they were simultaneously given 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 dosage of the polypeptide extract was calculated by converting it to the human dose of 300 mg / day, i.e., the rat dose was 31.2 mg / kg (calculation formula: 300 / 60 * 6.25). After raising each group of rats under the above conditions for 8 weeks, blood glucose levels and glycated hemoglobin (HbA1c) were examined. The results are shown in Table 4 below.

[0044] As can be seen from the results in Table 4, the blood glucose levels of the polypeptide group rats were clearly lower than those of the blank group rats, with a decrease of approximately 38.8%. Furthermore, the glycated hemoglobin levels of the polypeptide group rats were lower than those of the blank group rats, with a decrease of approximately 14.3%. These results indicate that when an individual has type 2 diabetes or is at high risk of developing type 2 diabetes, administering the polypeptide extract disclosed in this invention can effectively control the individual's blood glucose levels, thereby achieving the effect of preventing or delaying the onset of diabetes or glycemic imbalance-related diseases.

[0045] Table 4: Blood glucose levels and glycated hemoglobin levels in each rat group after the experiment. JPEG0007880154000004.jpg16147

[0046] Example 4: Animal testing (3)

[0047] Several 8-week-old SD rats were prepared and randomly divided into two groups. The blank group rats were given only high-calorie feed, while the polypeptide group rats were given both high-calorie feed and the polypeptide extract described in Example 1. The dosage of the polypeptide extract was calculated by converting it to the human dose of 300 mg / day, i.e., the rat dose was 31.2 mg / kg (calculation formula: 300 / 60 * 6.25).

[0048] After raising rats from each group under the conditions described above for 8 weeks, their body weight was measured, and after sacrificing, the weight of their abdominal fat was measured. The results are shown in Table 5 below.

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

[0050] These results demonstrate that when an individual continues to consume a high-fat or high-calorie diet while simultaneously being administered the polypeptide extract disclosed in this invention, it is possible not only to suppress fat accumulation but also to increase fat metabolism, thereby achieving effects such as weight loss, reduction of body fat, or prevention of significant weight gain. In other words, the polypeptide extract disclosed in this invention can be used for the prevention and / or treatment of fat metabolism-related diseases such as obesity and metabolic syndrome.

[0051] Table 5: Post-test body weight and abdominal fat of each rat group JPEG0007880154000005.jpg16147

[0052] Example 5: Cell Test

[0053] As can be seen from the results of the aforementioned example, the polypeptide extract disclosed in Example 1 has activities such as lowering glycated hemoglobin, controlling blood glucose, and increasing fat metabolism. Therefore, polypeptides indicated by SEQ ID No.:1 to SEQ ID No.:5 were isolated from it, their sequences were determined, and they were also synthesized artificially, and the following cell tests were performed.

[0054] Mouse myoblast cell line C2C12 was prepared, divided into groups, and cultured for 24 hours. Then, human insulin (MedChemExpress), polypeptide 1 (SEQ ID No.:1), polypeptide 2 (SEQ ID No.:2), polypeptide 3 (SEQ ID No.:3), polypeptide 4 (SEQ ID No.:4), and polypeptide 5 (amino acid sequence code SEQ ID No.:5) as a control were added to the culture medium, and the final concentration of the medium was adjusted to 20 μL / mL. Each cell group was then cultured for a further 24 hours. After removing the culture medium from each cell group and washing with phosphate buffer, the phosphatase inhibitor RIPA buffer was added, and the mixture was allowed to react under refrigeration for 30 minutes. After thawing, extraction was performed by shaking with an ultrasonic oscillator for 5 minutes, and the supernatant of the nuclear cell group was obtained by centrifugation. Further analysis by Western blotting was performed 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 the expression of proteins related to blood glucose control, insulin receptor activation, and fatty acid metabolism in each cell group. JPEG0007880154000006.jpg77151

[0056] As can be seen from the results in Table 6, polypeptide 1 with amino acid sequence SEQ ID No.:1, polypeptide 2 with amino acid sequence SEQ ID No.:2, and polypeptide 3 with amino acid sequence SEQ ID No.:3 disclosed in the present invention were able to promote insulin receptor phosphorylation without insulin induction, 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 fatty acid synthesis pathway. On the other hand, polypeptide 4 with 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 it was also able to promote insulin receptor phosphorylation without insulin induction, increase the expression level of GLUT4, and improve the expression levels of PGC-1α and PPARα, which are important proteins in the fatty acid synthesis pathway. Polypeptide 5, with amino acid sequence SEQ ID No.:5, failed to improve the expression level of phosphorylated AKT compared to the polypeptides with amino acid sequences SEQ ID No.:1 to SEQ ID No.:4, and also showed poor improvement in the expression levels of GLUT4, PGC-1α, and PPARα. As can be seen from the results above, each polypeptide with amino acid sequences SEQ ID No.:1 to SEQ ID No.:4 disclosed in this invention was superior to insulin in terms of 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 polypeptide with amino acid sequences SEQ ID No.:1 to SEQ ID No.:4 disclosed in the present invention can activate insulin receptors, stimulate cells to actively transport glucose and control blood glucose, and promote cellular fatty acid metabolism, and can be used to achieve blood glucose control and weight reduction effects in individuals. In other words, by administering an effective amount of each polypeptide with amino acid sequences SEQ ID No.:1 to SEQ ID No.:4 disclosed in the present invention, or a composition containing any of the above polypeptides, to an individual, it is possible to achieve the treatment and / or prevention of blood glucose imbalance or lipid metabolism disorder-related diseases such as diabetes, hyperglycemia, obesity, and metabolic syndrome.

Claims

1. A composition for controlling blood glucose, comprising a polypeptide selected from the group consisting of the amino acid sequences indicated by SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No.

4.

2. The composition according to claim 1, wherein the composition comprises polypeptides represented by SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, and SEQ ID No. 4.

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