Herring polypeptide with hemoglobin-lowering and blood sugar-lowering effects and preparation method thereof

Abstract

The present invention discloses a herring polypeptide with hemoglobin-lowering and blood sugar-lowering effects, and a preparation method thereof. The amino acid sequence of the polypeptide is Gly-Pro-Ala-Gly-Asp-Pro-Ala or Pro-Pro-Val-Glu-Glu-Pro. The present invention is the first to isolate and obtain two DPP-IV inhibitory polypeptides from Baltic herring. These polypeptides have the functions of improving insulin resistance, lowering blood sugar and glycated hemoglobin levels, lowering insulin levels, inhibiting DPP-IV activity, lowering plasma C-peptide and GLP-1 levels, and reducing the expression of hepatic inflammatory mediators. These polypeptides can be used as drugs to provide a certain preventive or auxiliary treatment effect for people with chronic metabolic disorders such as type 2 diabetes, and have very broad application prospects.

Description

Technical Field

[0001] This invention relates to a herring polypeptide with glycated hemoglobin-lowering and blood glucose-lowering effects and its preparation method, belonging to the pharmaceutical field. Background Technology

[0002] Globally, 10% of adults have diabetes, a figure that has increased by 16% in two years, reaching 537 million adults worldwide in 2021. An estimated 44.7% of adults with diabetes (240 million people) remain undiagnosed, with over four-fifths (81%) living in low- or middle-income countries. The continued rise in diabetes prevalence is primarily due to the surge in type 2 diabetes and related risk factors, including obesity, unhealthy diets, and widespread lack of physical activity. Globally, diabetes accounts for 12.2% of all deaths, with nearly half of these deaths occurring in people under 60 years of age. As a chronic metabolic disease, diabetes increases the risk of early death.

[0003] Diabetes mellitus is mainly classified into three types: type 1, type 2, and gestational diabetes mellitus. Type 2 diabetes mellitus (T2DM) accounts for approximately 90%–95% of all cases. T2DM is a complex endocrine and metabolic disease caused by insulin resistance or the inability of peripheral tissues to absorb glucose, leading to the body's inability to fully utilize insulin. T2DM has multiple causes, and glucagon-like peptide-1 (GLP-1) / dipeptidyl peptidase-IV (DPP-IV) inhibitors have emerged as a novel treatment approach. GLP-1 can stimulate rapid insulin release, stimulate insulin biosynthesis, inhibit β-cell apoptosis, and promote β-cell differentiation in vivo. However, GLP-1 can be rapidly cleaved and inactivated by DPP-IV. Therefore, DPP-IV inhibitors, which inhibit DPP-IV activity without degrading GLP-1, have become one of the main research directions for the treatment of T2DM. Currently, the most extensively researched and clinically applied DPP-IV inhibitors include sitagliptin, vildagliptin, and saxagliptin. However, these chemically synthesized drugs have certain toxic side effects. Protein peptides derived from natural proteins with DPP-IV inhibitory activity have attracted widespread attention due to their high safety for consumption, strong tolerability, and lack of toxic side effects.

[0004] Baltic herring is the primary fish caught in the Baltic Sea fisheries. However, its value remains low due to low processing rates, as the majority is used as livestock feed. Furthermore, only about 4% of the catch is consumed as food. It is estimated that the value of Baltic herring could increase by approximately 17.9 times if it could be processed into high-value-added products, such as health supplements. Therefore, Baltic herring is an inexpensive and sustainable source of bioactive peptides with potential commercial applications in functional foods, nutritional products, and pharmaceuticals.

[0005] Fish protein hydrolysates have been reported to contain peptides that inhibit DPP-IV activity. For example, the gelatin peptides Gly-Pro-Ala-Glu and Gly-Pro-Gly-Ala from Atlantic salmon skin have been reported to have DPP-IV inhibitory activity. Animal model studies have shown that protein hydrolysates from fish such as Atlantic salmon, cod, blue cod, halibut, tilapia, and Atlantic herring have beneficial effects on glucose homeostasis. Furthermore, cod muscle protein and blue cod hydrolysates can protect obese Zuckerfa / fa rats from the development of diabetic nephropathy. In a double-blind crossover study of healthy volunteers, consumption of cod protein hydrolysates (20 mg / kg) reduced serum glucose and GLP-1 concentrations in subjects. Therefore, developing functional peptides with hypoglycemic activity using Baltic herring protein has significant economic and social benefits. It can be used as a food supplement to enhance the value of Baltic herring protein, as a functional ingredient in health foods, and can also be applied to the production of next-generation antidiabetic drugs, showing great promise. Summary of the Invention

[0006] Objectives of the Invention: The first objective of this invention is to provide a polypeptide with hypoglycemic activity. The second objective of this invention is to provide a method for preparing the aforementioned polypeptide. The third objective of this invention is to provide applications of the aforementioned polypeptide.

[0007] Technical solution: A polypeptide of the present invention, wherein the amino acid sequence of the polypeptide is Gly-Pro-Ala-Gly-Asp-Pro-Ala or Pro-Pro-Val-Glu-Glu-Pro.

[0008] Furthermore, the polypeptide was isolated from Baltic herring.

[0009] The method for preparing the polypeptide of the present invention includes the following steps:

[0010] (1) Grind Baltic herring into a paste, mix with water, stir, and heat to 50-65°C to form a crude protein solution;

[0011] (2) Add alkaline protease and metal ion-containing compounds to the crude protein solution for a first enzymatic hydrolysis, and then add flavor protease for a second enzymatic hydrolysis to obtain the hydrolysate.

[0012] (3) After inactivating the enzyme in the enzymatic hydrolysate, centrifuge at low temperature to remove the precipitate, concentrate the supernatant and freeze dry;

[0013] (4) The concentrated supernatant was filtered, and extracts of <3kDa, 3-10kDa and >10kDa were collected respectively and purified by reversed-phase liquid chromatography.

[0014] (5) The information obtained in step (4) is compared, screened and predicted with the Atlantic herring sequence, and the DPP-Ⅳ inhibitory activity is predicted. The sequence of the polypeptide is determined based on the structural characteristics of the DPP-Ⅳ inhibitory peptide.

[0015] (6) Detect the in vitro inhibitory activity of DPP-Ⅳ peptides with high predicted DPP-Ⅳ inhibitory activity obtained in step (5), and use a hyperglycemic animal model to detect their hypoglycemic activity, thereby screening out peptide compounds that truly have in vivo hypoglycemic activity.

[0016] Further, in step (2), the concentration of the crude protein solution added during the first enzymatic hydrolysis is 1%–4%, the pH is 7.0–8.0, the concentration of the alkaline protease added is 1%–4%, the concentration of metal ions is 0.1–2.0 mmol / L, the hydrolysis temperature is 50–65℃, and the hydrolysis time is 2–4 hours; the metal ions include one or more of zinc ions, magnesium ions, or calcium ions; in step (2), the concentration of the flavor protease added during the second enzymatic hydrolysis is 1%–3%, the hydrolysis temperature is 50–65℃, and the hydrolysis time is 1–2 hours.

[0017] Further, the metal ion-containing compound mentioned in step (2) is one or more of 0.1-1.2 mmol / L ZnSO4, 0.1-1.2 mmol / L LmnSO4 and 0.1-2.0 mmol / L CaCl2.

[0018] Furthermore, in step (3), the enzyme inactivation temperature is 80–95°C and the time is 10–30 minutes; the centrifugation temperature is 4°C and the conditions are centrifugation at 10000g for 15–25 minutes.

[0019] Further, the specific steps of step (5) are as follows: (5.1) Import the data obtained in step (4) into ProggenesisQI for proteomics data analysis. The imported data is a search for Atlantic herring (37,476 sequence materials). Since the Baltic herring sequence database is currently unavailable, Atlantic herring is used as a close relative. The low-intensity threshold and high-intensity threshold are set to 135V and 30V, respectively. For identification, non-specific enzyme digestion is applied to deamidate asparagine or glutamine, hydroxylate aspartic acid, lysine, asparagine and proline, oxidize methionine, and perform variable modifications on glutamine or glutamic acid to form N-pyroglutamic acid. The quality tolerance of peptides is 10 ppm, and the fragment tolerance is 20 ppm. The false discovery FDR is 1%. To improve identification, peptides with a score <5 are removed, thereby reducing the absolute quality error to >20 ppm. (5.2) All peptides identified in step (5.1) were subjected to computer-based DPP-IV prediction (http: / / camt.pythonanywhere.com / iDPPIV-SCM). Peptides with a score >294 were considered to have potential DPP-IV inhibitory activity. Potential DPP-IV peptides with more than 10 amino acids were removed. The top 100 peptides from the remaining peptides were selected based on their relative abundance from Proggenesis. Peptides containing trypsin cleavage sites (lysine or arginine) or chymotrypsin cleavage sites (phenylalanine, tyrosine, tryptophan, or cysteine ​​residues) were removed. The sequences of the peptides were determined by sequence interpretation using Masslynx software, alignment to the Baltic herring protein database, and structural features of DPP-IV inhibitory peptides.

[0020] Furthermore, the in vitro inhibitory activity assay for DPP-Ⅳ in step (6) is as follows:

[0021] Forty 6-week-old male SPF C57BL / 6J mice were housed at a room temperature of 22–24°C and humidity of 40–60%, with 12-hour / 12-hour alternating day and night cycles, and free access to food and water for one week. Afterward, they were randomly divided into 5 groups of 8 mice each: a low-fat group, a high-fat, high-sugar model group, a PPVEEP active peptide intervention group, a GPAGDPA active peptide intervention group, and a sitagliptin positive control group. The low-fat diet contained 4% (w / w) fat and 0% (w / w) fructose; the high-fat, high-sugar diet contained 22% (w / w) fat and 23% (w / w) fructose.

[0022] The experiment lasted 16 weeks: Animals were acclimatized for one week before the experiment began. Treatments for each group were as follows: 1) Low-fat group: fed a low-fat diet with free access to water. 2) High-fat, high-sugar model group: fed a high-fat, high-sugar diet with free access to water. 3) PPVEEP active peptide intervention group: fed a high-fat, high-sugar diet with free access to water containing 0.6 mg / mL PPVEEP (peptide sequence: Pro-Pro-Val-Glu-Glu-Pro). 4) GPAGDPA active peptide intervention group: fed a high-fat, high-sugar diet with free access to water containing 0.6 mg / mL GPAGDPA (peptide sequence: Gly-Pro-Ala-Gly-Asp-Pro-Ala). 5) Sitagliptin positive control group: fed a high-fat, high-sugar diet with free access to water containing 0.1 mg / mL sitagliptin.

[0023] Subsequently, changes in blood glucose and plasma glycated hemoglobin levels, plasma insulin levels, insulin tolerance, plasma DPP-IV activity, plasma C-peptide, plasma GLP-1 levels, and liver inflammatory mediator expression in mice were examined.

[0024] The application of the polypeptide of the present invention in at least one of the following a to f:

[0025] (a) To prepare products that improve insulin resistance;

[0026] (b) To prepare products that lower blood glucose and glycated hemoglobin levels;

[0027] (c) Prepare products that lower insulin levels;

[0028] (d) Prepare products that inhibit the activity of DPP-Ⅳ;

[0029] (e) Prepare products that reduce plasma C-peptide and GLP-1 levels;

[0030] (f) Prepare products that reduce the expression of liver inflammatory mediators.

[0031] Furthermore, the product includes pharmaceuticals.

[0032] The application of the polypeptide of the present invention in the preparation of drugs for the prevention or treatment of type 2 diabetes.

[0033] The application of the polypeptide of the present invention in the preparation of DPP-Ⅳ inhibitors.

[0034] Furthermore, in the above applications, the concentration of the polypeptide is above 0.6 mg / mL.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: The present invention is the first to isolate two DPP-IV inhibitory polypeptides from Baltic herring, which have the functions of improving insulin resistance, reducing blood glucose and glycated hemoglobin levels, reducing insulin levels, inhibiting DPP-IV activity, reducing plasma C-peptide and GLP-1 levels, and reducing the expression of liver inflammatory mediators. They can be used as drugs to have a certain preventive or adjunctive therapeutic effect on people with chronic metabolic disorders such as type 2 diabetes, and have a very broad application prospect. Attached Figure Description

[0036] Figure 1 In vitro DPP-IV activity inhibition rate of potential hypoglycemic peptides;

[0037] Figure 2 : Glucose tolerance and insulin tolerance in different groups of mice (* and *** indicate statistically significant differences compared with the low-fat group, respectively, P<0.05 and P<0.001); # , ## and ### This indicates statistically significant differences compared to the high-fat, high-sugar model group (P<0.05, P<0.01, and P<0.001).

[0038] Figure 3 Insulin resistance index of mice in different groups (*** indicates statistical difference compared with low-fat group, P<0.001); ### This indicates a statistically significant difference (P < 0.001) compared to the high-fat, high-sugar model group.

[0039] Figure 4 Fasting blood glucose and glycated hemoglobin levels in different groups of mice (* and *** indicate statistically significant differences compared to the low-fat group, P<0.05 and P<0.001, respectively; # and *** indicate statistically significant differences compared to the low-fat group). ## This indicates statistically significant differences compared to the high-fat, high-sugar model group (P<0.05 and P<0.01, respectively).

[0040] Figure 5 Fasting insulin levels in mice in different groups (*** indicates a statistically significant difference compared to the low-fat group, P<0.001); ### This indicates a statistically significant difference (P < 0.001) compared to the high-fat, high-sugar model group.

[0041] Figure 6 Plasma DPP-Ⅳ activity levels in different groups of mice (* indicates a statistically significant difference compared to the low-fat group, P<0.05); # This indicates a statistically significant difference (P<0.05) compared to the high-fat, high-sugar model group.

[0042] Figure 7 Plasma C-peptide and GLP-1 levels in mice from different groups (* indicates a statistically significant difference compared to the low-fat group, P<0.05); # This indicates a statistically significant difference (P<0.05) compared to the high-fat, high-sugar model group.

[0043] Figure 8 : Expression levels of liver inflammatory mediators in different groups of mice (*** indicates statistically significant differences compared with the low-fat group, P<0.001); # , ## and ### This indicates statistically significant differences compared to the high-fat, high-sugar model group (P<0.05, P<0.01, and P<0.001, respectively). Detailed Implementation

[0044] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0045] Example 1: Preparation of polypeptides

[0046] (1) Extraction of active peptides

[0047] Baltic herring from Turku, Finland, was ground using a meat grinder. The fish paste was then mixed with water (1:1, w / w), stirred, and heated to 55°C to form a crude protein solution. The crude protein solution was adjusted to pH 7.5 using alkaline phosphatase buffer, and 2% (w / w) alkaline protease was added, along with 0.6 mmol / L ZnSO4, 0.8 mmol / L MnSO4, and 1.0 mmol / L CaCl2. The mixture was stirred at 55°C for 2 hours for enzymatic hydrolysis, followed by the addition of 2% (w / w) flavor protease and further enzymatic hydrolysis at 60°C for 1 hour. After the reaction, the enzymes were inactivated at 85°C for 15 minutes. The mixture was centrifuged at 10,000g for 15 minutes at 4°C, filtered to remove the precipitate, concentrated, and then freeze-dried.

[0048] (2) Separation and purification

[0049] The above lyophilized sample was dissolved in deionized water to prepare a concentration of 10 mg / mL. After filtration through a 0.22 μm filter membrane, ultrafiltration was performed using 3 kDa and 10 kDa molecular weight ultrafiltration centrifuge tubes. The fractions <3 kDa, 3-10 kDa, and >10 kDa were collected and lyophilized for later use.

[0050] The separated components were dissolved in 1 mL (0.1%, v / v) formic acid and filtered using an Amicon ultrafiltration membrane with a molecular weight cutoff (MWCO) of 10 kDa to remove macromolecules. 300 μL of the filtrate was collected, purified using a SepPakt C18 (Waters, Milford, MA) filtration system, and eluted with 20% (v / v) acetonitrile. The eluted peptide mixture was evaporated to dryness under a nitrogen stream and then lyophilized. The dried peptide mixture was then reconstituted in 200 μL of a solution containing 0.1% (v / v) formic acid and 5% (v / v) acetonitrile, and subsequently separated at 45 °C using an Acquity UPLC HSS C18 column (1.8 μm, 2.1 × 150 mm, Waters) with the following parameters: flow rate 0.4 mL / min, separation gradient 5%–35%. The buffer consisted of 20.0% acetonitrile and 0.1% formic acid. Subsequently, liquid chromatography coupled with a Synapt G2 Si Q-Tof mass spectrometer (Waters, Milford MA) was used, and mass spectrometry data were acquired in positive ion mode using a data-independent method within the mass range of m / z 50–2000 at a spectral acquisition time of 0.5 s. Throughout the run, leucine enkephalin at m / z 556.2771 was monitored and locked for mass correction.

[0051] (3) Sequence identification and activity detection

[0052] The obtained mass spectrometry data were imported into Proggenesis QI for proteomics data analysis. The imported data was searched for Atlantic herring (37,476 sequence materials). Since the Baltic herring sequence database is currently unavailable, Atlantic herring (data source: National Library of Medicine: https: / / www.ncbi.nlm.nih.gov / ) was used as a close relative. Low-intensity thresholds and high-intensity thresholds were set to 135V and 30V, respectively. For identification, non-specific enzyme digestion was applied, followed by deamidation of asparagine or glutamine, hydroxylation of aspartic acid, lysine, asparagine, and proline, oxidation of methionine, and variable modification of glutamine or glutamate to form N-pyroglutamic acid. The mass tolerance for peptides was 10 ppm, and for fragments, it was 20 ppm. The false discovery rate (FDR) was 1%. To improve identification, peptides with a score <5 were removed, resulting in an absolute mass error >20 ppm.

[0053] Subsequently, all identified peptides underwent computer-based DPP-IV activity inhibition prediction (http: / / camt.pythonanywhere.com / iDPPIV-SCM). Peptides with a score >294 were considered to have potential DPP-IV inhibitory activity, while potential DPP-IV inhibitory peptides with more than 10 amino acids were removed. Based on the relative abundance from Proggenesis, the top 100 peptides from the remaining potentially active peptides were selected. Peptides containing trypsin cleavage sites (lysine or arginine) or chymotrypsin cleavage sites (phenylalanine, tyrosine, tryptophan, or cysteine ​​residues) were then removed. The sequences of the peptides were determined by sequence interpretation using Masslynx software (Waters, Milford, USA), alignment to the Baltic herring protein database, and structural characterization of DPP-IV inhibitory peptides. The results are shown in Tables 1 and 2, with 86 peptides identified as having DPP-IV inhibitory activity potential.

[0054] Table 1. Screening steps and identification results of DPP-IV activity-inhibiting peptides

[0055]

[0056] Table 2. Information on 86 peptides with potential DPP-IV inhibitory activity

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068] Example 2: Detection of in vitro DPP-IV activity inhibition of peptides

[0069] Four peptides with the highest predicted activity from Example 1 were selected for synthesis to further determine the inhibition of DPP-IV activity in vitro. The sequences were PPVEEP, GPAGDPA, HyP-HyP-GRPGF, and GADPEVIVS, respectively. These synthetic peptides were purchased from Biomatik (ON, Canada) and had a purity exceeding 95%.

[0070] The above four peptides were subjected to in vitro DPP-IV activity inhibition assay, and the specific procedures are as follows:

[0071] (1) Principle

[0072] DPP-IV inhibitors were screened using a chromogenic substrate method with Gly-Pro-PNA as the substrate. The detection principle of this method is that under alkaline conditions, DPP-IV catalyzes the hydrolysis of the substrate Gly-Pro-p-nitroanilide to generate yellow p-nitroaniline, which has a characteristic absorption peak at a wavelength of 405 nm. The absorbance measured at 405 nm by an enzyme-linked immunosorbent assay (ELISA) reader reflects the level of enzyme activity.

[0073] (2) Method

[0074] Samples: Dissolve the samples (PPVEEP, GPAGDPA, HyP-HyP-GRPGF and GADPEVIVS) in 100mM Tris-HCl buffer (pH=8.0) to prepare a 40mg / mL stock solution. Then dilute the stock solution to concentrations of 0μM, 100μM, 300μM, 500μM and 1000μM to prepare sample solutions.

[0075] Substrate: Gly-Pro-PNA solution, prepared as 1.59 mM Gly-Pro-PNA solution using 100 mM Tris-HCl buffer (pH = 8.0).

[0076] Enzyme: DPP-IV solution, prepared as 0.1 U DPP-IV solution using 100 mM Tris-HCl buffer (pH = 8.0).

[0077] Termination solution: 1M acetate-sodium acetate buffer (pH=4.0).

[0078] First, add the above-mentioned concentrations of sample (inhibitor) and substrate sequentially to a 96-well plate and incubate at 37°C for 10 minutes. Then, add 0.1 U of DPP-IV enzyme solution, mix well, and incubate at 37°C for 60 minutes. Finally, add 50 μL of 1M acetate-sodium acetate buffer as a stop solution to terminate the reaction. Measure the absorbance at 405 nm using a microplate reader (A). 405The total reaction volume was 150 μL, and the sample volumes for each group are shown in Table 3. The experiment was divided into four sample groups (PPVEEP, GPAGDPA, HyP-HyP-GRPGF, and GADPEVIVS), a sample blank group, a control group, and a blank group. Each group had three replicates. After adding the reactants as listed in the table, the final volume was made up to 150 μL with Tris-HCl buffer.

[0079] Table 3. Experimental groupings and sample dosages for DPP-VI inhibitory activity

[0080]

[0081]

[0082] Method for calculating the DPP-IV activity inhibition rate:

[0083]

[0084] The inhibition rate (%) versus peptide concentration was plotted using GraphPad Prism 5, and the IC50 value was calculated using a nonlinear regression [inhibitor] and a response-variable slope (four parameters) function. The results are as follows: Figure 1 As shown, the IC50 values ​​of the synthetic peptides PPVEEP and GPAGDPA for in vitro DPP-IV inhibition were 936 μM and 1049 μM, respectively. However, HyP-HyP-GRPGF and GADPEDVIVS, which contain hydroxyproline residues, showed no in vitro DPP-IV inhibitory activity.

[0085] Example 3 Performance Analysis of Active Peptides

[0086] The C57BL / 6J mice used in the following examples were purchased from Changzhou Slack Laboratory Animal Co., Ltd.; the standard diet (LFD, D12450B) and high-fat, high-sugar diet (HFD, D12451) used in the following examples were purchased from Nantong Trofi Feed Technology Co., Ltd. (Nantong, China); the insulin ELISA kit used in the following examples was purchased from Roch Diagnostic (Switzerland); the blood glucose and glycated hemoglobin assay kits used in the following examples were purchased from Nanjing Jiancheng Bioengineering Institute; the plasma C-peptide ELISA kit used in the following examples was purchased from Avivasysbio (USA); the plasma active glucagon-like peptide-1 (GLP-1) assay kit used in the following examples was purchased from Millipore (USA); the plasma DPP-IV activity assay kit used in the following examples was purchased from Enzo Inc. (USA); the plasma TNF-α ELISA kit used in the following examples was purchased from Invitrogen (USA); the IL-1β ELISA kit was purchased from Sigma-Aldrich (USA); and the IL-6 ELISA kit was purchased from BD. Biosciences, Inc. (USA); the liver NF-κB P65 and activating protein 1 ELISA kits used in the following examples were all purchased from Cayman Chemical, Inc. (USA); all total RNA extraction kits, reverse transcription kits, and real-time quantitative PCR kits were purchased from Takara Biotechnology, Inc. (Japan).

[0087] 3.1 Mouse rearing process

[0088] Forty 6-week-old male SPF C57BL / 6J mice were housed at a room temperature of 22–24°C and humidity of 40–60%, with 12-hour / 12-hour alternating day and night cycles, and free access to food and water for one week. Afterward, they were randomly divided into five groups of eight mice each: a low-fat group, a high-fat, high-sugar model group, an intervention group receiving PPVEEP (polypeptide sequence Pro-Pro-Val-Glu-Glu-Pro) bioactive peptides, an intervention group receiving GPAGDPA (polypeptide sequence Gly-Pro-Ala-Gly-Asp-Pro-Ala) bioactive peptides, and a sitagliptin positive control group. The low-fat diet contained 4% (w / w) fat and 0% (w / w) fructose; the high-fat, high-sugar diet contained 22% (w / w) fat and 23% (w / w) fructose.

[0089] The experiment lasted 16 weeks: Animals were acclimatized for one week before the experiment began. Treatments for each group were as follows: 1) Low-fat diet group: fed a low-fat diet with free access to water. 2) High-fat, high-sugar model group: fed a high-fat, high-sugar diet with free access to water. 3) PPVEEP active peptide intervention group: fed a high-fat, high-sugar diet with free access to water containing 0.6 mg / mL active peptide. 4) GPAGDPA active peptide intervention group: fed a high-fat, high-sugar diet with free access to water containing 0.6 mg / mL active peptide. 5) Sitagliptin positive control group: fed a high-fat, high-sugar diet with free access to water containing 0.1 mg / mL sitagliptin.

[0090] 3.1.1 Effects of bioactive peptides PPVEEP and GPAGDPA on insulin tolerance in hyperglycemic mice

[0091] Mice were fasted for 12 hours prior to the glucose tolerance test (GTT) on Monday of week 16, following conditions 3.1. Blood samples were collected from the tail of the mice after an injection of glucose (1 g / kg body weight, Sigma-Aldrich, USA). Blood glucose concentrations were measured at 0, 15, 30, 45, 60, 90, and 120 minutes post-injection using an Accu-Chek Advantage II blood glucose meter (Roche Diagnostic, Switzerland). GTT and ITT are expressed as area under the curve (AUC).

[0092] Prior to the insulin tolerance test (ITT) on Thursday of week 16, mice were fasted for 12 hours and injected with insulin (1.5 UI / kg body weight, Sigma-Aldriich, USA). Blood samples were collected from the tail of the mice. Blood insulin concentrations were measured at 0, 15, 30, 45, 60, 90, and 120 minutes post-injection using the aforementioned ELISA kit, following the kit manufacturer's instructions. GTT and ITT are expressed as area under the curve (AUC). Homeostasis model assessment of insulin resistance (HOMA-IR) scores was calculated using fasting blood glucose and insulin concentrations.

[0093] Depend on Figure 2 It was found that mice in the high-fat, high-glucose model group exhibited insulin resistance compared to the control group. Insulin resistance was improved in the PPVEEP active peptide group, GPAGDPA active peptide group, and sitagliptin positive control group compared to the high-fat, high-glucose model group. Figure 3 It can be seen that the insulin resistance index of mice in the high-fat and high-glucose model group was significantly higher than that of mice in the control group, while the insulin resistance index of mice in the PPVEEP active peptide group, GPAGDPA active peptide group, and sitagliptin positive control group was significantly lower than that of mice in the high-fat and high-glucose model group.

[0094] 3.1.2 Effects of bioactive peptides PPVEEP and GPAGDPA on blood glucose and glycated hemoglobin levels in hyperglycemic mice

[0095] After feeding according to conditions 3.1, blood was collected from mice. Blood samples were centrifuged at 3000 rpm for 10 minutes to obtain plasma, which was then stored at -80°C until biochemical analysis. Blood glucose concentration and glycated hemoglobin levels were measured using the kit according to the manufacturer's instructions. Figure 4 It can be seen that, compared with the control group, the blood glucose concentration and glycated hemoglobin level of mice in the high-fat and high-glucose model group were significantly increased. Compared with the high-fat and high-glucose model group, the blood glucose concentration and glycated hemoglobin level of mice in the PPVEEP active peptide group, GPAGDPA active peptide group and sitagliptin positive control group were significantly decreased.

[0096] 3.1.3 Effects of bioactive peptides PPVEEP and GPAGDPA on insulin levels in hyperglycemic mice

[0097] After feeding according to conditions 3.1, blood was collected from mice. Blood samples were centrifuged at 3000 rpm for 10 minutes to obtain plasma, which was then stored at -80°C until biochemical analysis. Plasma insulin levels were measured using the aforementioned ELISA kit, following the kit manufacturer's instructions. Figure 5 It can be seen that the plasma insulin concentration of mice in the high-fat and high-glucose model group was significantly higher than that of mice in the control group. The plasma insulin concentration of mice in the PPVEEP active peptide group, GPAGDPA active peptide group and sitagliptin positive control group was significantly lower than that of mice in the high-fat and high-glucose model group.

[0098] 3.1.4 Effects of bioactive peptides PPVEEP and GPAGDPA on DPP-IV activity in hyperglycemic mice

[0099] After feeding according to conditions 3.1, blood was collected from mice. Plasma samples were obtained by centrifugation at 3000 rpm for 10 minutes and stored at -80°C until biochemical analysis. DPP-IV activity was measured using the aforementioned kit according to the manufacturer's instructions. Figure 6 It can be seen that, compared with the control group, the plasma DPP-IV activity of mice in the high-fat and high-glucose model group was significantly increased, and the DPP-IV activity of mice in the PPVEEP active peptide group, GPAGDPA active peptide group and sitagliptin positive control group was significantly decreased compared with the high-fat and high-glucose model group.

[0100] 3.1.5 Effects of bioactive peptides PPVEEP and GPAGDPA on C-peptide and GLP-1 in hyperglycemic mice

[0101] After feeding according to conditions 3.1, blood was collected from mice. Blood samples were centrifuged at 3000 rpm for 10 minutes to obtain plasma, which was then stored at -80°C until biochemical analysis. Plasma C-peptide and GLP-1 concentrations were measured using the aforementioned kit according to the kit manufacturer's instructions. Figure 7 It was found that the plasma C-peptide concentration was significantly higher in the high-fat, high-glucose model group compared to the control group. The plasma C-peptide concentration was significantly lower in the PPVEEP active peptide group, GPAGDPA active peptide group, and sitagliptin positive control group compared to the high-fat, high-glucose model group. Furthermore, the plasma GLP-1 concentration was significantly higher in the high-fat, high-glucose model group compared to the control group. The plasma GLP-1 concentration was significantly lower in the GPAGDPA active peptide group compared to the high-fat, high-glucose model group, but not significantly lower in the PPVEEP active peptide group and the sitagliptin positive control group.

[0102] 3.1.6 Effects of bioactive peptides PPVEEP and GPAGDPA on the expression of inflammatory mediators in the liver of hyperglycemic mice

[0103] After feeding according to conditions 3.1, mice were euthanized, and the livers of each animal were removed and stored at -80°C until analysis. Gene expression levels of liver inflammatory mediators tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were measured using the aforementioned kits, following the manufacturer's instructions. Specifically, total RNA was extracted from liver tissue using Trizol reagent (Takara Biotechnology, Japan), and cDNA was synthesized from 2 μg of total RNA using the Prime Script RT kit (Takara Biotechnology, Japan). RT-qPCR was then performed using the SYBR Green Real-Time PCR Master Mix (Takara Biotechnology, Japan) on a CFX quantitative PCR system (Bio-Rad, USA). Primer sequences were synthesized by Generay Biotechnology (Shanghai, China), and the specific sequences are shown in Table 4. The relative expression of each target gene was normalized to a β-Actin threshold cycle (CT) value, and a comparison threshold cycle of 2 was used. -ΔΔCT The method is quantified.

[0104] Table 4. Primer Sequences

[0105]

[0106]

[0107] Depend on Figure 8It can be seen that, compared with the control group, the expression of inflammatory mediators in the liver of mice in the high-fat and high-sugar model group was significantly increased. Compared with the high-fat and high-sugar model group, the expression of inflammatory mediators in the liver of mice in the PPVEEP active peptide group, GPAGDPA active peptide group and sitagliptin positive control group was significantly decreased.

Claims

1. A polypeptide, characterized in that The amino acid sequence of the polypeptide is Gly-Pro-Ala-Gly-Asp-Pro-Ala or Pro-Pro-Val-Glu-Glu-Pro.

2. A method for preparing the polypeptide according to claim 1, characterized in that: The steps include: (1) Grind Baltic herring into minced fish, mix with water, stir, and heat to 50-65°C to form a crude protein solution; (2) adding alkaline protease and a metal ion-containing compound to the crude protein solution for primary enzymatic hydrolysis, and then adding flavor protease for secondary enzymatic hydrolysis to obtain an enzymatic hydrolyzate; (3) inactivating the enzyme in the hydrolyzate and centrifuging at low temperature to remove the precipitate, concentrating the supernatant and freeze-drying it; (4) Filter the concentrated supernatant to collect extracts of <3 kDa, 3-10 kDa, and >10 kDa, respectively, and purify them by reversed-phase liquid chromatography; (5) comparing the sequence information obtained in step (4) with the Atlantic herring sequence, screening, and predicting the DPP-IV inhibitory activity, and determining the sequence of the polypeptide based on the structural characteristics of the DPP-IV inhibitory peptide; (6) Detecting the DPP-IV inhibitory activity in vitro of the polypeptide with a high predicted value of DPP-IV inhibitory activity screened in step (5), and detecting its hypoglycemic activity using a hyperglycemic animal model to screen out polypeptide compounds that truly have hypoglycemic activity in vivo.

3. The preparation method according to claim 2, characterized in that The primary enzymatic hydrolysis crude protein solution in step (2) is added at a mass concentration of 1% to 4%, the pH is 7.0 to 8.0, the alkaline protease is added at a mass concentration of 1% to 4%, the metal ion concentration is 0.1 to 2.0 mmol / L, the enzymatic hydrolysis temperature is 50 to 65° C., and the enzymatic hydrolysis time is 2 to 4 hours; the metal ions include one or more of zinc ions, magnesium ions, or calcium ions; the secondary enzymatic hydrolysis flavor protease in step (2) is added at a mass concentration of 1% to 3%, the enzymatic hydrolysis temperature is 50 to 65° C., and the enzymatic hydrolysis time is 1 to 2 hours.

4. The preparation method according to claim 2, characterized in that The metal ion-containing compound in step (2) is one or more of 0.1-1.2 mmol / L ZnSO4, 0.1-1.2 mmol / L MnSO4 and 0.1-2.0 mmol / L CaCl2.

5. The preparation method according to claim 2, characterized in that The enzyme inactivation temperature in step (3) is 80-95°C for 10-30 minutes; the centrifugation temperature is 4°C and the centrifugation condition is 10,000 g for 15-25 minutes.

6. Use of the polypeptide according to claim 1 in at least one of the following a to c: (a) preparing a product for improving insulin resistance; (b) preparing products that lower blood sugar and glycated hemoglobin levels; (c) preparing a product that reduces insulin levels.

7. The use according to claim 6, characterized in that The products include pharmaceuticals.

8. Use of the polypeptide according to claim 1 in preparing a drug for preventing or treating type 2 diabetes.

9. The use according to any one of claims 6 to 7 or 8, characterized in that: The concentration of the polypeptide in the application is above 0.6 mg / mL.

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