Bioactive peptides derived from prolamine and their breakdown products

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a bioactive peptide derived from millet prolamin and a decomposition product thereof, wherein the amino acid sequence of the bioactive peptide is shown as SEQ ID NO:1, and the length of the decomposition product is 2-6 amino acids. The bioactive peptide and the decomposition product thereof have a significant blood glucose-lowering effect and can enhance the sensitivity of cells to insulin. In addition, the bioactive peptide and the decomposition product thereof have good biological safety, small molecular weight, easy absorption, non-toxicity and other characteristics, and can be used for developing drugs for treating diabetes and its complications, and have a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to bioactive peptides and their degradation products. Background Technology

[0002] Millet, a grain with a long history, is a high-quality source of plant protein, with protein content accounting for about 9.7% of its dry weight. It is commonly used to supplement people's daily protein needs. In addition, millet is rich in carbohydrates, protein, dietary fiber, B vitamins, various minerals, and antioxidants such as flavonoids. Therefore, millet has a variety of health benefits, including anti-oxidation, blood sugar regulation, digestion promotion, sleep improvement, and cardiovascular protection. Millet protein can be processed through enzymatic hydrolysis to obtain small molecule peptides. Some of these peptides and their decomposition products have specific biological activities, such as anti-oxidation and blood sugar control. However, there are still issues such as the unclear amino acid sequence and efficacy of characteristic peptides in millet.

[0003] Therefore, screening out millet peptides and their decomposition products with biological activity and clear efficacy is of great significance for the application of millet protein products in the development of pharmaceuticals and other fields. Summary of the Invention

[0004] This invention aims to at least partially address one of the technical problems existing in the prior art. To this end, this invention provides bioactive peptides derived from millet prolysin and their degradation products. The bioactive peptides and their degradation products of this invention have significant hypoglycemic effects and can enhance cellular sensitivity to insulin. Furthermore, these bioactive peptides and their degradation products possess good biocompatibility, small molecular weight, easy absorption, and non-toxicity, and can be used in the development of drugs for the treatment of diabetes and its complications, showing broad application prospects.

[0005] In a first aspect, the present invention provides a bioactive peptide and its degradation products. According to embodiments of the present invention, the amino acid sequence of the bioactive peptide is as shown in SEQ ID NO: 1, and the length of the degradation products is 2-6 amino acids. The bioactive peptide and its degradation products according to embodiments of the present invention have significant hypoglycemic effects and can enhance cellular sensitivity to insulin. Because they also possess advantages such as good biocompatibility, small molecular weight, and easy absorption, they can be applied to the development of drugs for the treatment of diabetes and its complications, and have broad application prospects.

[0006] According to embodiments of the present invention, the above-mentioned bioactive peptides and their degradation products may also have the following additional technical features: According to an embodiment of the present invention, the amino acid sequence of the decomposition product is selected from at least one of the following: SEQ ID NO: 2 and FFV.

[0007] In a second aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the bioactive peptide and its degradation products described in the first aspect. The nucleic acid molecule according to embodiments of the present invention enables efficient expression of the aforementioned bioactive peptide or its degradation products, facilitates large-scale industrial production of the bioactive peptide and its degradation products, and provides convenience for the subsequent development and application of drugs for the treatment of diabetes and its complications.

[0008] In a third aspect, the present invention provides a construct. According to embodiments of the invention, it includes the nucleic acid molecule described in the second aspect. The construct according to embodiments of the invention enables the aforementioned nucleic acid molecule to be efficiently expressed in host cells, thereby achieving stable and efficient production of the aforementioned bioactive peptide or its degradation products.

[0009] In a fourth aspect, the present invention provides a recombinant cell. According to embodiments of the invention, it comprises the nucleic acid molecule described in the second aspect or the construct described in the third aspect. The recombinant cell according to embodiments of the invention can efficiently express the aforementioned bioactive peptides or their degradation products under suitable conditions, thereby enabling their large-scale industrial production.

[0010] In a fifth aspect, the invention proposes the use of the bioactive peptides and their degradation products described in the first aspect, the nucleic acid molecules described in the second aspect, the constructs described in the third aspect, or the recombinant cells described in the fourth aspect in the preparation of pharmaceutical products. According to embodiments of the invention, the pharmaceutical product has at least one of the following uses: prevention, relief, adjunctive treatment, or treatment of diabetes; and control of blood glucose levels.

[0011] Those skilled in the art will understand that the features and advantages described above for bioactive peptides and their degradation products, nucleic acid molecules, constructs or recombinant cells also apply to this application, and will not be repeated here.

[0012] In a sixth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises one or more of the following: the bioactive peptide and its degradation products as described in the first aspect, the nucleic acid molecule as described in the second aspect, the construct as described in the third aspect, and the recombinant cell as described in the fourth aspect.

[0013] Those skilled in the art will understand that the features and advantages described above for bioactive peptides and their degradation products, nucleic acid molecules, constructs or recombinant cells also apply to this use, and will not be repeated here.

[0014] According to embodiments of the present invention, the above-described pharmaceutical composition may further have the following additional technical features: According to an embodiment of the present invention, the pharmaceutical composition further comprises: pharmaceutically acceptable excipients.

[0015] In a seventh aspect, the present invention provides a method for improving cellular sensitivity to insulin. According to an embodiment of the invention, the method includes contacting cells with the bioactive peptides and their degradation products described in the first aspect; wherein the cells are cells obtained through in vitro culture. The method according to the embodiments of the invention, by contacting cells with bioactive peptides and their degradation products, can significantly improve cellular sensitivity to insulin while having minimal impact on cell activity, providing a platform for developing drugs to treat diabetes and its complications.

[0016] According to embodiments of the present invention, the above method may further have the following additional technical features: According to embodiments of the present invention, the cells include one or more of skeletal muscle cells, liver cancer cells, hepatocytes, and adipocytes.

[0017] According to an embodiment of the present invention, the cells are liver cancer cells, and the liver cancer cells are HepG2 cells.

[0018] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0019] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a mass spectrometry result of the bioactive peptide FFVGGNWK sample in Example 1 of the present invention; Figure 2 This is a graph showing the results of the oral glucose tolerance test in mice in Example 2 of this invention, where # represents... P <0.05, ## is P <0.01 (NCD vs HFD), * indicates P <0.05 (HFD vs FFVGGNWK), A is the result of the glucose tolerance test for each group of mice, and B is the result of the calculated area under the blood glucose curve in the glucose tolerance test for each group of mice; Figure 3 This is a mass spectrometry result of the bioactive peptide FFV sample in Example 3 of the present invention; Figure 4 This is a mass spectrometry result of the bioactive peptide GGNW sample in Example 3 of the present invention; Figure 5This is a graph showing the results of measuring cell viability and glucose consumption of HepG2 cells at different insulin concentrations in Example 3 of the present invention. * indicates... P <0.05, A is the result of cell viability measurement of HepG2 cells under different insulin concentrations, and B is the result of glucose consumption measurement of HepG2 cells under different insulin concentrations; Figure 6 This is a graph showing the results of cell viability and glucose consumption of HepG2 cells under different treatment conditions in Example 3 of the present invention, where * indicates... P <0.05, A is the result of glucose consumption measurement of HepG2 cells under different treatment conditions, and B is the result of cell viability measurement of HepG2 cells under different treatment conditions. Detailed Implementation

[0020] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0021] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0022] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0023] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0024] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0025] In this article, the term "OGTT" refers to the oral glucose tolerance test, a method used to detect the function of pancreatic β cells and the body's ability to regulate blood glucose. It is mainly used to diagnose diabetes, determine the type of diabetes, and understand the body's glucose metabolism. This method can simulate postprandial hyperglycemia. When insulin resistance occurs in the body, the sensitivity of insulin target tissues to insulin decreases, and the uptake, conversion, and utilization of glucose by peripheral tissues (including liver, fat, and skeletal muscle) decreases, thereby increasing blood glucose levels, which is manifested as impaired glucose tolerance.

[0026] Bioactive peptides and their degradation products This invention proposes a bioactive peptide and its degradation products. According to embodiments of the invention, the amino acid sequence of the bioactive peptide is shown in SEQ ID NO: 1, and the degradation products have a length of 2-6 amino acids. The bioactive peptide and its degradation products according to embodiments of the invention exhibit significant hypoglycemic effects and enhance cellular sensitivity to insulin. Due to their good biocompatibility, small molecular weight, and easy absorption, they can be applied to the development of drugs for the treatment of diabetes and its complications, showing broad application prospects.

[0027] According to an embodiment of the present invention, the amino acid sequence of the bioactive peptide is specifically as follows: FFVGGNWK (SEQ ID NO: 1) According to embodiments of the present invention, the amino acid sequence of the degradation product is selected from at least one of the following: SEQ ID NO: 2 and FFV. This further clarifies the amino acid sequence of the bioactive peptide degradation product.

[0028] It should be noted that the amino acid sequences mentioned in this invention are all shown in the order from N-terminus to C-terminus.

[0029] In this article, the term "decomposition product" refers to small molecule peptide fragments obtained from bioactive peptides through enzymatic hydrolysis or other methods. Some decomposition products retain similar biological activity to the original bioactive peptides.

[0030] Nucleic acid molecules This invention proposes a nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes the aforementioned bioactive peptide and its degradation products. The nucleic acid molecule according to embodiments of the invention enables efficient expression of the aforementioned bioactive peptide or its degradation products, facilitates large-scale industrial production of the bioactive peptide and its degradation products, and provides convenience for the subsequent development and application of drugs for the treatment of diabetes and its complications.

[0031] It should be noted that those skilled in the art should understand that the nucleic acid molecules mentioned herein actually include any one or both of the complementary double strands; although in most cases only one strand is given, the other complementary strand is actually disclosed as well; in addition, the nucleic acid molecule sequences in this invention include DNA or RNA forms, and disclosing one of them means that the other is also disclosed.

[0032] Construct This invention proposes a construct. According to embodiments of the invention, it includes the aforementioned nucleic acid molecule. The construct according to embodiments of the invention enables the aforementioned nucleic acid molecule to be efficiently expressed in host cells, thereby achieving stable and efficient production of the aforementioned bioactive peptide or its degradation products.

[0033] Exemplarily, the construct may be a vector; it should be noted that, for the purposes of this document, a vector refers to a nucleic acid molecule capable of self-replication within a suitable host, which transfers the inserted nucleic acid molecule to host cells and / or between host cells; the vector may include vectors primarily used for inserting DNA or RNA into cells, vectors primarily used for replicating DNA or RNA, and expression vectors primarily used for transcription and / or translation of DNA or RNA; the vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell; by culturing a suitable host cell containing the vector, the vector may produce the aforementioned bioactive peptides and their degradation products, the vector including viral vectors, plasmids, bacteriophages, etc.

[0034] Recombinant cells This invention proposes a recombinant cell. According to embodiments of the invention, it includes the aforementioned nucleic acid molecules or the aforementioned constructs. The recombinant cell according to embodiments of the invention can efficiently express the aforementioned bioactive peptides or their degradation products under suitable conditions, thereby enabling their large-scale industrial production.

[0035] Applications in preparation or pharmaceuticals This invention proposes the use of the aforementioned bioactive peptides and their degradation products, the aforementioned nucleic acid molecules, the aforementioned constructs, or the aforementioned recombinant cells in the preparation of pharmaceuticals. According to embodiments of the invention, the pharmaceutical product has at least one of the following uses: prevention, relief, adjunctive treatment, or treatment of diabetes; and control of blood glucose levels.

[0036] Those skilled in the art will understand that the features and advantages described above for bioactive peptides and their degradation products, nucleic acid molecules, constructs or recombinant cells also apply to this application, and will not be repeated here.

[0037] Pharmaceutical Composition This invention provides a pharmaceutical composition. According to embodiments of the invention, the pharmaceutical composition comprises one or more of the aforementioned bioactive peptides and their degradation products, the aforementioned nucleic acid molecules, the aforementioned constructs, and the aforementioned recombinant cells.

[0038] Those skilled in the art will understand that the features and advantages described above for bioactive peptides and their degradation products, nucleic acid molecules, constructs or recombinant cells also apply to this use, and will not be repeated here.

[0039] According to embodiments of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable excipients. Thus, the addition of pharmaceutical excipients allows the pharmaceutical composition to better exert the efficacy of the aforementioned bioactive peptides or their degradation products.

[0040] Exemplary excipients include excipients, diluents, stabilizers, etc.; it should be noted that pharmaceutically acceptable excipients may include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a specific target dosage form. Except for any conventional excipients that are incompatible with the functional components in the pharmaceutical composition of the present invention, such as any adverse biological effects or harmful interactions with any other components of the pharmaceutically acceptable composition, their use is also within the scope of the present invention.

[0041] Methods to improve cellular sensitivity to insulin This invention proposes a method for improving cellular sensitivity to insulin. According to an embodiment of the invention, the method includes contacting cells with the aforementioned bioactive peptides and their degradation products; wherein the cells are cells obtained through in vitro culture. The method according to the embodiment of the invention, by contacting cells with bioactive peptides and their degradation products, can significantly improve cellular sensitivity to insulin while having minimal impact on cell activity, providing a platform for developing drugs to treat diabetes and its complications.

[0042] According to embodiments of the present invention, the cells include one or more of skeletal muscle cells, liver cancer cells, hepatocytes, and adipocytes. Therefore, the method of the present invention has broad applicability, allowing for the selection of suitable cells based on different application scenarios and experimental objectives.

[0043] It should be noted that the method of improving cell sensitivity to insulin of the present invention is not only applicable to the skeletal muscle cells and other cells specifically listed above, but also applicable to any other cell type that requires insulin and can be obtained through in vitro culture. All cell types shown are within the protection scope of the present invention.

[0044] According to an embodiment of the present invention, the cells are liver cancer cells, specifically HepG2 cells. Therefore, the method of the present invention is applicable to HepG2 cells and can be used in the development of therapeutic drugs for diabetes in liver cancer patients.

[0045] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0046] Example 1: Preparation of bioactive peptide FFVGGNWK 1. Extraction of millet prolysin Defatted millet flour was uniformly dispersed in 70% ethanol solution at a ratio of 1:7 (w / v) to obtain a defatted millet flour sample solution. The defatted millet flour sample solution was then reacted in a water bath at 37℃ with shaking for 4 h, followed by centrifugation at 8000 rpm for 15 min and collection of the supernatant. The supernatant was then dialyzed for 36 h using a 3.5 kDa dialysis bag (Shanghai Yuanye Biotechnology Co., Ltd.), during which the deionized water was replaced 4-5 times to obtain the dialysate. The dialysate was then centrifuged at 7000 rpm for 5 min and the precipitate was collected to obtain millet prolysin sample, which was lyophilized and stored at -20℃ for later use.

[0047] 2. Enzymatic hydrolysis of millet prolysin The millet protein sample obtained in step 1 was uniformly dispersed in deionized water at a ratio of 3:100 (w / v) to obtain a millet protein sample solution. The millet protein sample solution was then pretreated with ultrasonic power at 400 W for 20 min, followed by treatment with alkaline protease (Sigma, catalog number P5380) at pH=9.0 and 55℃ for 2 h to obtain an enzymatic hydrolysate. The enzymatic hydrolysate was then heated at 90℃ for 15 min to inactivate the enzyme and stop hydrolysis. After the enzymatic hydrolysate cooled to room temperature, it was centrifuged at 6000 rpm for 20 min, the supernatant was collected and lyophilized to obtain the millet protein hydrolysate.

[0048] 3. Isolation, purification, and sequence identification of polypeptides The millet prolysin hydrolysate obtained in step 2 was subjected to peptide separation, purification, and sequence identification. A bioactive peptide was identified, and the amino acid sequence of the bioactive peptide is shown in SEQ ID NO: 1.

[0049] FFVGGNWK (SEQ ID NO: 1) Subsequently, Hefei Peptide Library Biotechnology Co., Ltd. was commissioned to prepare the bioactive peptide FFVGGNWK sample using solid-phase synthesis.

[0050] Mass spectrometry results of the bioactive peptide FFVGGNWK sample are shown below. Figure 1 .

[0051] The results showed that the obtained bioactive peptide FFVGGNWK sample was verified by mass spectrometry and had a purity of over 95%.

[0052] Example 2: Hypoglycemic effect of bioactive peptide FFVGGNWK 1. Grouping of experimental animals Thirty male C57BL / 6J mice (4 weeks old, 18 ± 2 g) were acclimatized for one week and then randomly divided into three groups: a normal control group (NCD, n=10), a diabetic mouse model group (HFD, n=10), and a group treated with bioactive peptide FFVGGNWK sample + diabetic mouse model group (FFVGGNWK, n=10). The treatment steps for each group of mice are as follows: Normal control group (NCD): After being fed a standard diet for 4 weeks, mice were injected intraperitoneally with 90 mg / kg body weight of citrate buffer (pH=4.5) once in week 5. Starting from week 6, mice were administered 200 mg / kg body weight of physiological saline by gavage for 5 weeks to obtain control mice. Diabetic mouse model group (HFD): After being fed a high-fat diet for 4 weeks, streptozotocin (0.1 mol / L citrate buffer, pH=4.5) was injected intraperitoneally once in week 5. Starting from week 6, physiological saline was administered by gavage for 5 weeks to obtain diabetic mouse models. Bioactive peptide FFVGGNWK sample treatment + diabetic mouse model group (FFVGGNWK): After feeding a high-fat diet for 4 weeks, streptozotocin (0.1 mol / L citrate buffer, pH=4.5) was injected intraperitoneally once in week 5. Starting from week 6, bioactive peptide FFVGGNWK sample solution was administered by gavage for 5 weeks to obtain bioactive peptide intervention mice.

[0053] 2. Oral glucose tolerance test The control mice, diabetic model mice, and bioactive peptide-treated mice obtained in step 1 were subjected to an oral glucose tolerance test (OGTT). The specific steps are as follows: After fasting for 12 hours, mice in each group were administered glucose solution (1 g / kg) by gavage according to their body weight. Blood glucose was measured in the tail vein at 0 min, 15 min, 30 min, 60 min, 90 min and 120 min after gavage, and blood glucose-time curves were plotted. The area under the blood glucose curve (AUC) is calculated according to Formula 1.

[0054] AUC = (0.5 × T0 + T30) / 2 + (1.5 × T60 + T120) / 2 (Formula 1) Among them, T0, T30, T60 and T120 represent the blood glucose levels at 0 min, 30 min, 60 min and 120 min after glucose injection, respectively.

[0055] The results of the oral glucose tolerance test in each group of mice are shown in the figure. Figure 2 .

[0056] The results showed that the blood glucose level of the control mice remained stable, dropping to 9.95 mmol / L at 30 min and then returning to normal at 120 min. In contrast, the blood glucose level of the diabetic model mice peaked at 31.46 mmol / L at 30 min and then gradually decreased. However, the blood glucose level of this group of mice was always higher than that of the control mice and the bioactive peptide-treated mice. In addition, compared with the diabetic model mice, the AUC value of the bioactive peptide-treated mice was significantly reduced by 16.01%.

[0057] The above results indicate that the bioactive peptide FFVGGNWK can effectively improve impaired glucose tolerance in diabetic mice and regulate their postprandial hyperglycemia levels.

[0058] Example 3: In vivo stability study of bioactive peptide FFVGGNWK 1. Decomposition products of bioactive peptide FFVGGNWK Normal C57BL / 6J mice were fasted overnight, and the bioactive peptide FFVGGNWK sample obtained in Example 1 was administered by gavage at a dose of 10 mL / kg. At 1 h, 2 h, and 4 h, one mouse was anesthetized with isoflurane and sacrificed by cervical dislocation to obtain blood samples. The blood samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0059] The results showed that the two peptides FFV and GGNW (SEQ ID NO: 2) obtained by the decomposition of the bioactive peptide FFVGGNWK were stable in the blood.

[0060] Subsequently, Hefei Peptide Library Biotechnology Co., Ltd. was commissioned to prepare bioactive peptide FFV and bioactive peptide GGNW samples using solid-phase synthesis.

[0061] Mass spectrometry results of bioactive peptide FFV sample are shown below. Figure 3 The mass spectrometry results of the bioactive peptide GGNW sample are shown in [the table below]. Figure 4 .

[0062] The results showed that the obtained bioactive peptide FFV and bioactive peptide GGNW samples were both verified by mass spectrometry and had a purity of over 95%.

[0063] 2. The hypoglycemic effect of bioactive peptide FFVGGNWK and its decomposition products (1) Establishment of an insulin-resistant HepG2 cell model HepG2 cells (purchased from the cell bank of Peking Union Medical College) were cultured in high-glucose DMEM medium (purchased from Gibco, Inc., USA, catalog number C11965500BT) supplemented with 10% (v / v) fetal bovine serum (purchased from Henan Miracle Biotechnology Co., Ltd., catalog number MRC-C23011-A) and 1% penicillin-streptomycin. After reaching a cell density of 80%–90% at 37°C and 5% CO2, the HepG2 cells were washed with phosphate buffer, dispersed with trypsin, and transferred to new culture flasks.

[0064] Then HepG2 cells (1*10) were used. 4 After inoculating cells / mL into 96-well plates and culturing for 24 h, the medium was replaced with serum-free high-glucose DMEM (purchased from Gibco, USA, catalog number C11965500BT) for starvation treatment for 6 h, and then maintained at different insulin concentrations (0 μmol / L, 1*10). -5 μmol / L, 1*10 -6 μmol / L, 1*10 -7 μmol / L, 1*10 -8 μmol / L, 1*10 -9 HepG2 cells were incubated with insulin at different concentrations (μmol / L, 100 μL) for 24 h. Then, the cell viability of HepG2 cells at different insulin concentrations was determined by the CCK-8 assay, and the glucose consumption of HepG2 cells at different insulin concentrations was evaluated using the GOD-POD kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number S0201S).

[0065] The results of cell viability and glucose consumption of HepG2 cells at different insulin concentrations are shown in the figure. Figure 5 .

[0066] The results showed that when the insulin concentration was 1*10 -5 μmol / L ~ 1*10 -9 Within the μmol / L range, the cell viability of HepG2 cells remained above 95% ( Figure 5 In the above (A), insulin had no toxic effect on HepG2 cells within this concentration range; compared with HepG2 cells treated with insulin concentration (0 μmol / L), the glucose consumption of HepG2 cells showed a trend of first decreasing and then increasing with increasing insulin intervention concentration. Specifically, when the insulin concentration was 1*10... -6 At μmol / L, HepG2 cells exhibited the highest glucose consumption ( Figure 5 (B in the middle).

[0067] The above results indicate that selecting 1*10 -6 A μmol / L insulin concentration was used to establish an insulin-resistant HepG2 cell model.

[0068] (2) Toxicity test and glucose consumption analysis Referring to the cell model construction process in step (1), follow the 1*10 -6 An insulin-resistant HepG2 cell model was established using an insulin concentration of μmol / L, resulting in the HepG2 model group (model group, insulin-intervened); simultaneously, a HepG2 control group (normal group, no insulin intervention) was obtained using an insulin concentration of 0 μmol / L.

[0069] Then, HepG2 model cells were incubated with peptide solutions of different concentrations (25 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL, and 800 μg / mL) for 24 h. The cell viability and glucose consumption of HepG2 cells under different treatment conditions were evaluated using the CCK-8 assay and the GOD-POD kit. The peptide solutions included bioactive peptide FFVGGNWK sample solution (FFVGGNWK group), bioactive peptide FFV sample solution (FFV group), and bioactive peptide GGNW sample solution (GGNW group).

[0070] The results of cell viability and glucose consumption of HepG2 cells under different treatment conditions are shown in the figure. Figure 6 .

[0071] The results show: (1) Compared with the HepG2 control group cells, the glucose consumption of HepG2 model group cells gradually increased with the increase of peptide concentration. Specifically, when the peptide concentration increased from 25 μg / mL to 800 μg / mL, the glucose consumption of GGNW group increased from 2.68 mg / mL to 3.92 mg / mL, the glucose consumption of FFVGGNWK group increased from 3.05 mg / mL to 3.99 mg / mL, and the glucose consumption of FFV group increased from 2.60 mg / mL to 3.80 mg / mL. All three peptides showed hypoglycemic effects. Overall, the bioactive peptide FFVGGNWK sample solution had the best hypoglycemic effect, followed by the bioactive peptide GGNW sample solution, and then the bioactive peptide FFV sample solution. Figure 6 (A in the middle) (2) Compared with the HepG2 control group cells, the cell viability of the HepG2 model group cells gradually decreased with the increase of peptide concentration. Among them, when the concentration of bioactive peptide GGNW sample solution, bioactive peptide FFVGGNWK sample solution and bioactive peptide FFV sample solution reached 800 μg / mL, the cell viability was 86.85%, 94.51% and 86.76% respectively, and their cell viability was still greater than 80%. Overall, the bioactive peptide FFVGGNWK sample solution had the least impact on the cell viability of HepG2 cells. Figure 6 (B in the middle).

[0072] The above results indicate that bioactive peptides FFVGGNWK, FFV, and GGNW all exhibit good hypoglycemic effects on HepG2 cells and have no toxic effects. Among them, bioactive peptide FFVGGNWK has the best hypoglycemic effect.

[0073] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0074] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The use of a bioactive peptide, a nucleic acid molecule encoding the bioactive peptide, a construct comprising the nucleic acid molecule, or a recombinant cell expressing the bioactive peptide in the preparation of a pharmaceutical product, characterized in that, The drug has the following uses: Prevention, relief, adjunctive treatment, or treatment of diabetes; The amino acid sequence of the bioactive peptide is shown in SEQ ID NO:

1.

2. A method for increasing cellular glucose consumption in vitro, characterized in that, include: Contacting cells with bioactive peptides; The cells in question are insulin-resistant liver cancer cells obtained through in vitro culture. The amino acid sequence of the bioactive peptide is shown in SEQ ID NO:

1.

3. The method according to claim 2, characterized in that, The liver cancer cells were HepG2 cells.

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