Dry-cured ham-derived oligopeptides and use thereof

Abstract

The application discloses a dry-cured ham source oligopeptide and application thereof. The dry-cured ham source oligopeptide comprises at least one of the following amino acid sequences: ELIDQDARDLY, YHEHRSDLN, YKATEPVIAF, INKVEELKKKY and GEKLKRQKY. The dry-cured ham source oligopeptide is applied to preparation of a drug for treating a glycolipid metabolism disorder disease. The extracted and verified oligopeptide can be specifically combined with an AdipoR2 receptor, has stronger glycolipid metabolism regulation activity than a KRQKYD polypeptide, can significantly improve various glycolipid metabolism disorder diseases, and has a good application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to a medicinal polypeptide, in particular to a dry-cured ham-derived oligopeptide and its application in the preparation of a medicine. BACKGROUND

[0002] Glycolipid metabolism disorder is a chronic disease caused by multiple factors, and its initial feature is the accumulation of lipids in hepatocytes. In addition, glycolipid metabolism disorder is also a key factor for inducing metabolic syndrome such as diabetes, obesity, fatty liver and atherosclerosis. The pathogenesis of glycolipid metabolism disorder is closely related to insulin resistance, liver fat synthesis, liver lipid peroxidation, oxidative stress, expression of obesity-related cytokines and intestinal microbial disorder. Fat accumulation and degeneration are the main characteristics of glycolipid metabolism disorder, so maintaining glycolipid metabolism homeostasis may be one of the effective strategies for preventing and treating glycolipid metabolism disorder. At present, there is a lack of therapeutic drugs for glycolipid metabolism disorder, so it is urgent to develop effective prevention strategies. Since glycolipid metabolism disorder is closely related to diet, dietary intervention is considered to be an effective strategy for preventing glycolipid metabolism disorder.

[0003] Food-derived bioactive peptides refer to functional peptides taken by organisms from outside the body, which are directly or indirectly derived from food proteins and generally exist in dietary proteins with specific amino acid sequences. Food-derived peptides are a very safe and easy-to-industrialize polypeptide, so they are particularly concerned in the fields of nutrition and food science. At present, the development of polypeptides containing functional ingredients is an innovative strategy for functional foods, health products and drugs. A large amount of food-derived proteins and waste generated during processing are usually used as fertilizers or directly discharged, which not only causes resource waste but also causes environmental pollution.

[0004] During the maturation period of fermented ham, a large amount of proteins are hydrolyzed into amino acids and small molecule peptides, which are enriched in the final product. These small molecule peptides have the function of scavenging free radicals in the human body and preventing various chronic diseases. At present, there is no report on screening and extracting dry-cured ham-derived oligopeptides based on AdipoR2 receptors to improve glycolipid metabolism disorder from fermented ham to solve the problems of low content of functional factors, difficult extraction and low efficiency in the application of dietary intervention of glycolipid metabolism disorder. SUMMARY

[0005] The purpose of the present application is to provide a dry-cured ham-derived oligopeptide to solve the problem of how to efficiently improve glycolipid metabolism disorder by taking AdipoR2 receptor as the polypeptide action target. Another purpose of the present application is to provide an application of the dry-cured ham-derived oligopeptide in the preparation of a drug for treating glycolipid metabolism disorder to solve the problem of how to prepare the drug for treating glycolipid metabolism disorder.

[0006] Technical scheme: The dry-cured ham-derived oligopeptide provided by the present application comprises at least one of the following amino acid sequences:

[0007] ELIDQDARDLY; YHEHRSDLN; YKATEPVIAF; INKVEELKKKY; GEKLKRQKY.

[0008] Preferably, the dry-cured ham-derived oligopeptide contains the following amino acid sequence:

[0009] ELIDQDARDLY and / or YHEHRSDLN.

[0010] The second aspect of the present application discloses the use of the above dry-cured ham-derived oligopeptide in the preparation of a drug for treating a glycolipid metabolism disorder.

[0011] Preferably, the glycolipid metabolism disorder comprises at least one of insulin resistance, hyperglycemia, diabetes, hyperlipidemia, and fatty liver.

[0012] Preferably, the content of the dry-cured ham-derived oligopeptide in the drug is 1-200 mg / mL. The drug can selectively add a pharmaceutically acceptable adjuvant according to the needs of different preparations.

[0013] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages:

[0014] The extracted and verified oligopeptide of the present application can specifically bind to the AdipoR2 receptor, and exhibits stronger glycolipid metabolism regulation activity than the existing KRQKYD polypeptide, can significantly improve various glycolipid metabolism disorder diseases, and has good prospects for application in both medicine and food. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 High-performance liquid chromatogram of the target oligopeptide powder;

[0016] Figure 2 Secondary mass spectrum of the target oligopeptide A-E;

[0017] Figure 3 Effect of the five oligopeptides on lipid deposition of hepatocytes in a non-alcoholic fatty liver (NAFLD) cell model;

[0018] Figure 4 Effect of D peptide and E peptide on body weight and organ index of NAFLD mice; wherein, (A) is the body weight change amount; (B) is the body weight change amount; (C) is the liver coefficient; (D) is the white fat coefficient.

[0019] Figure 5Effects of D peptide and E peptide on lipid accumulation in NAFDL mice; wherein, (A) is a representative image of fat H&E staining, scale bar, 50 μm; (B) is a graph of serum total cholesterol level; (C) is a graph of serum triglyceride level; (D) is a graph of serum low-density lipoprotein cholesterol level; (E) is a graph of serum high-density lipoprotein cholesterol level; (F) is a graph of free fatty acid level.

[0020] Figure 6 Effects of D peptide and E peptide on lipid deposition in the liver of NAFDL mice; wherein, (A) is a representative image of liver H&E and oil red O staining, scale bar, 50 μm; (B) is a graph of liver TC content; (C) is a graph of liver TG content; (D) is a graph of liver HDL-C content; (E) is a graph of liver LDL-C content; (F) is a graph of serum ALT activity; (G) is a graph of serum AST activity.

[0021] Figure 7 Effects of D peptide and E peptide on oxidative stress in the liver of NAFDL mice; wherein, (A) is a graph of liver SOD activity; (B) is a graph of liver GSH-PX activity; (C) is a graph of liver CAT activity; (D) is a graph of liver MDA level.

[0022] Figure 8 Effects of D peptide and E peptide on insulin resistance in NAFDL mice; wherein, (A) is a graph of fasting blood glucose level of each group after 14 weeks of oligopeptide intervention; (B) is a graph of serum insulin level; (C) is a graph of oral glucose tolerance test (OGTT) blood glucose level; (D) is a graph of area under the curve; (E) is a graph of QUICK index; (F) is a graph of HOMA-IR index.

[0023] Figure 9 Regulatory effects of D peptide and E peptide on blood glucose metabolism signaling pathways in NAFDL mice.

[0024] Figure 10 Effects of D peptide and E peptide intervention on the AMPK / PPAR-α signaling pathway in the liver of NAFDL mice; wherein, (A) is a graph of Western blot detection of the expression level of each protein; (B) is a quantitative analysis of p-AMPK / AMPK; (C) is a quantitative analysis of PPAR-α; (D) is a quantitative analysis of CPT-1; (D) is a quantitative analysis of SREBP-1. DETAILED DESCRIPTION

[0025] The technical solutions of the present application will be further described below in combination with the drawings.

[0026] Example 1: A dry-cured ham-derived oligopeptide, the sequence of which is as follows:

[0027] Peptide A: YKATEPVIAF;

[0028] B-peptide: INKVEELKKKY;

[0029] C-peptide: GEKLKRQKY;

[0030] D-peptide: ELIDQDARDLY;

[0031] E-peptide: YHEHRSDLN.

[0032] The above five polypeptides were all extracted from dried cured ham, and the extraction methods are as follows:

[0033] (1) Defatting: Select fermented and matured dry-cured ham, remove visible fat and connective tissue, take the lean meat part and cut it into 0.5cm³ small pieces, mix the meat sample and n-hexane at a ratio of 1:3 (g / mL), extract twice at 30℃ with shaking, each time for 30min, centrifuge at 4000r / min for 10min, discard the supernatant, and vacuum dry the residue to remove residual solvent;

[0034] (2) Desalting: The defatted meat residue obtained in step 1 was dialyzed with flowing deionized water at 4°C for 24 hours to remove small molecule salts through a dialysis bag (molecular weight cutoff: 500 Da), and then freeze-dried and pulverized for later use.

[0035] (3) Oligopeptide extraction: The lyophilized powder obtained in step (2) was mixed with 0.1 mol / L hydrochloric acid solution at a ratio of 1:4 (g / mL), and homogenized in an ice-water bath (speed: 12000 rpm, number of homogenizations: 4 times, 1 min each time). The homogenized liquid was then sonicated for 1 h (ultrasonic power: 300 W, temperature: 30 ℃, pulse time: 30 s / 30 s). The liquid was filtered through 4 layers of gauze, and the filtrate was centrifuged at 8000 rpm for 20 min. The supernatant was then taken for later use.

[0036] (4) Decolorization: Add 0.1%-0.3% activated clay to the supernatant obtained in step (3), place it in an ice water bath and stir for 15 minutes (stirring speed: 80 rpm), then centrifuge at 4000 rpm for 5 minutes and take the supernatant for later use.

[0037] (5) Separation by fractionation: The supernatant obtained in step (4) is separated by gradient separation through hollow fiber columns with different molecular weight cutoffs (3000 Da, 500 Da). The fraction between 500-3000 Da is collected and freeze-dried to obtain crude peptide powder.

[0038] (6) Structure-directed purification:

[0039] Assembly and polymerization of molecularly imprinted polymer (MIP): 4-vinylpyridine (4-VP), 3-aminophenylboronic acid (APBA), methacrylic acid (MAA), acrylamide (AAm), ethylene glycol dimethacrylate (EGDMA) were mixed in a solvent system (acetonitrile: water = 7:3 v / v) at a ratio of n(APBA): n(4-VP): n(MAA): n(AAm): n(EGDMA) = 2:3:1.5:1:20, the assembly temperature was 4°C, the assembly time was 12h, and the molar ratio of template peptide (sequence KRQKY) to MIP monomer was 1:7.5. Subsequently, a thermal-initiated free radical polymerization reaction was carried out by adding 2,2'-azobisisobutyronitrile (0.5% w / v) under nitrogen protection at 60°C, the polymerization reaction was maintained at 60°C for 24h, and then annealed at 4°C for 12h to form a rigid skeleton and a fine recognition cavity, followed by the addition of a porogen (toluene:dodecanol = 3:1 v / v) to form a porous structure; the template peptide in the MIP pores was eluted using a Soxhlet extractor, 0.1% TFA / acetonitrile solution was added to destroy the hydrogen bond and π-π stacking interaction between the template peptide and the MIP, and the strong solubility of acetonitrile was used to assist the dissolution of the template peptide, followed by ultrasonic-assisted elution (ultrasonic function 300W, temperature 40°C), 300W power to avoid MIP structure damage, 40°C control within the polymer heat-resistant range. Subsequently, pH gradient elution (pH 2.0→10.0) was carried out to regulate the dissociation of borate ester bonds and other reversible covalent bonds, and the template peptide was completely eluted. Finally, the freeze-dried product was a molecularly imprinted polymer based on the triple recognition mechanism of tyrosine residues.

[0040] The crude peptide powder obtained in step (5) was configured into a 100mg / mL crude peptide solution with PBS buffer (containing 0.1M NaCl) at pH 7.4, purified by a medium-pressure glass chromatography column (inner diameter 10mm, length 100mm, wet packing 1g molecularly imprinted polymer, column efficiency: theoretical plate number ≥8000 / m) at a flow rate of 0.5mL / min, the filtrate was collected, followed by elution with 10CV deionized water to remove salt, and then elution with 5CV 10% acetonitrile to remove non-specific adsorption impurities, the filtrate was combined and discarded, and then eluted with borax buffer (containing 10% acetonitrile) at pH 9.5 at a flow rate of 1.0mL / min, the eluate was collected, dialyzed in a dialysis bag (molecular weight cut-off: 500Da) at 4°C for 24h using flowing deionized water to remove borax, and then freeze-dried to obtain the target oligopeptide powder.

[0041] (7) Mass spectrometry detection method: an UltiMate 3000 ultra-high performance liquid chromatography (UPLC) system combined with a Q-Exactive electrospray ionization mass spectrometer (Thermo Fisher Scientific) was used for chromatographic analysis of the oligopeptide in negative ion mode. The specific steps are as follows: the target oligopeptide powder was dissolved in high-performance liquid chromatography grade methanol to prepare a standard solution with a concentration of 100 μg / mL. Chromatographic column: Agilent Zorbax SB-C18 column (1.9 μm, 2.1 x 100 mm); column temperature: 35°C; flow rate: 0.2 mL / min; mobile phase: A phase is acidified water containing 0.1% formic acid, B phase is acetonitrile; injection volume: 2.0 μL; gradient elution program: 0-2 min, B phase remains 10%; 2-20 min, B phase linearly increases from 10% to 30%. The temperature is set to 550°C, and the spray voltage is set to 5500V (positive ion mode). After data acquisition, the converted data is processed for peak finding and peak alignment using MSDIAL ver4.6 software.

[0042] The results are shown in Figure 1 and Figure 2 Figure 1 A\B\C\D\E in the above-mentioned results are the peaks of A-E peptides, Figure 2 The mass spectrometry data of A-E peptides from top to bottom are shown in the above-mentioned results. As can be seen from Figure 1 and Figure 2 , the target oligopeptide powder contains at least five polypeptides of A-E peptides, and each of the five polypeptides has a tyrosine residue at the N- or C-terminus.

[0043] MIP was incubated with different concentrations of target peptides (including control peptide KRQKY and test peptides YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) in a buffer solution and placed in a constant temperature shaker for adsorption equilibrium. Then, MIP (solid phase) and supernatant (liquid phase) were separated by filtration. Peptide concentration determination: determined by high performance liquid chromatography (HPLC) quantitative method.

[0044] ​The calculation method of the saturation binding capacity is as follows: draw the binding isotherm (plot the amount of binding B against the free peptide concentration L). When L is high enough, B reaches a plateau, and this plateau value is the saturation binding capacity (mg / g MIP). The high saturation binding capacity of KRQKY in the table directly explains its advantage as a template peptide - the MIP has more binding sites customized for it and a higher degree of matching. But the saturation binding amount of the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) has also exceeded 70% of the template peptide, indicating that the MIP is feasible for the separation and screening of the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN).

[0045] The calculation method of the dissociation constant Kd is as follows: the Langmuir adsorption model is used to fit the binding isotherm, and the formula is: The dissociation constant Kd (unit: μM) is obtained by fitting the curve through nonlinear regression analysis (Origin software). The smaller the dissociation constant Kd, the stronger the binding affinity of the MIP to the target peptide. The dissociation constants of the test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) in the table are slightly larger than those of the template peptide, but the difference is not large, indicating that the MIP still has strong binding ability to the target peptide.

[0046] The calculation formula of the selectivity coefficient a is as follows: the selectivity coefficient a: a > 1: the binding affinity of the test peptide to the MIP is weaker than that of the control peptide KRQKY; all a in the table are > 1, indicating that the binding ability of the test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) to the MIP is slightly weaker than that of the template peptide KRQKY, but the difference is not large, indicating that the MIP still has strong selectivity for the target peptide. The results are shown in Table 1:

[0047] Table 1 Analysis of the saturation binding capacity, dissociation constant and selectivity coefficient of the target oligopeptide and IMP

[0048]

[0049] Example 2: To investigate the effect of the A-E peptides identified in Example 1 on the lipid deposition of hepatocytes in a non-alcoholic fatty liver (NAFLD) cell model, the experimental method is as follows:

[0050] Take the HepG2 cells in the logarithmic growth phase, digest them with 0.25% trypsin, and then dilute them to 5x105 Cells / well are inoculated into a 6-well plate (or 2x10 4 Cells / well are inoculated into a 24-well plate according to the detection requirements, 2 mL of DMEM medium containing 10% FBS is added, and the cells are cultured at 37°C in a 5% CO2 incubator for 24 hours to allow the cells to adhere and fuse to 70-80%. Discard the original culture medium, gently wash the cells with PBS twice (avoid damaging the adherent cells). Model group (HFD group): add serum-free DMEM medium containing 1 mM mixed fatty acid (PA:OA=1:2) + 1% BSA, and return to the incubator for continued culture for 24 hours. Control group (ND group): add serum-free DMEM medium containing 1% BSA, and return to the incubator for continued culture for 24 hours. Experimental group (A-E group): add serum-free DMEM medium containing 4 μM oligopeptide (A, B, C, D, E peptide) + 1 mM mixed fatty acid (PA:OA=1:2) + 1% BSA, respectively, and return to the incubator for continued culture for 24 hours. Collect the cells, wash with PBS, and add cell lysis solution (containing PMSF to inhibit protease), and ultrasonic lysis on ice (power 200W, 3 seconds / time, interval 5 seconds, total 5 times). Centrifuge at 12000 rpm for 10 minutes, take the supernatant, and measure the content of each index in the liver cells according to the TG, TC, HDL-C and LDL-C detection kit instructions.

[0051] The experimental results are shown in Figure 3 Among the five polypeptides, only D peptide and E peptide have a significant inhibitory effect on lipid deposition in liver cells, while A-C peptides have no such effect.

[0052] Example 3: Detection of the efficacy of D peptide and E peptide in improving glycolipid metabolism disorder in Example 1, the experimental method is as follows:

[0053] (1) 70 male C57BL / 6 mice (4 weeks old, body weight 18-20 grams) were purchased from Beijing Sbi Bio-technology Co., Ltd., and the animal qualification certificate number was SCXK (Tianjin) 2022-006. All animals were raised in an environment with controlled temperature (21±2°C) and relative humidity (60±10%), with a 12-hour light-dark cycle, and free access to food and water. All experimental operations strictly followed the "Regulations on the Management of Experimental Animals in China", were conducted under the guidance of the "Guide to the Care and Use of Laboratory Animals", and were approved by the Animal Research Ethics Committee of Chuzhou University.

[0054] (2) All mice were acclimatized in an SPF-grade laboratory for 7 days, and then randomly divided into 6 groups (n=8 per group). The specific grouping and treatment are as follows: Control group (ND group): given basal diet and gavage with an equal volume of physiological saline; D-peptide and E-peptide combined control group (OP group): given basal diet + D-peptide and E-peptide mixed in a 1:1 mass ratio by gavage (400 mg / kg / d); Model group (HFD group): fed high-fat diet (formula: 61.8% basal diet, 20% lard, 3% cholesterol, 0.2% bile salts, 15% egg yolk powder) and gavage with an equal volume of physiological saline; Oligopeptide D group (D group): high-fat diet + oligopeptide D by gavage (200 mg / kg / d); Oligopeptide E group (E group): high-fat diet + oligopeptide E by gavage (200 mg / kg / d). The basal diet and high-fat diet were provided by Nanjing Shengmin Co., Ltd. The gavage was performed continuously for 14 weeks, once a day. Food intake, water consumption, and body weight of mice in each group were monitored weekly. In the final week, all mice were fasted overnight, euthanized with CO2, and their eyes were enucleated for blood collection and serum separation. Liver, colon, and pancreas tissues were collected for biochemical and histopathological analysis; heart, kidney, spleen, and white adipose tissue (WAT, including perirenal fat, epididymal fat, and mesenteric fat) were collected and weighed, and organ index (organ weight / body weight) was calculated. Cecal tissue was preserved in… 80°C, used for subsequent microbiome analysis of cecal contents.

[0055] like Figure 4 As shown in Figures A and B, a high-fat diet for 14 weeks significantly increased the body weight of NAFLD model mice (HFD group), by 25.49% compared to the normal diet group (ND group). Intervention with D-peptide and E-peptide significantly inhibited the abnormal weight gain induced by the high-fat diet; the body weight of the oligopeptide D group and oligopeptide E group decreased by 8.40% and 12.29% respectively compared to the HFD group. Notably, compared to the ND group, the body weight of mice in the OP-intervention normal diet group (OP group) did not decrease significantly. This indicates that D-peptide and E-peptide can inhibit the abnormal weight gain induced by a high-fat diet without affecting the normal weight gain during development in normal mice, and have no physiological toxicity. To preliminarily assess the liver pathological changes in NAFLD mice and the protective effect of oligopeptides on the liver, organ coefficients (liver coefficient and fat coefficient) were evaluated, and the results are as follows: Figure 4 As shown in Figure C, the liver coefficient of the HFD group mice was significantly higher than that of the ND group by 11.53%, and decreased by 6.51% and 7.42% respectively after intervention with oligopeptide D and oligopeptide E groups. Meanwhile, a high-fat diet induced an increase in the fat coefficient of NAFLD mice, while oligopeptide intervention effectively antagonized this abnormal change. Figure 4The fat coefficient of the mice in the oligopeptide intervention groups (oligopeptide D group and oligopeptide E group) was significantly reduced by 15.37% and 23.41% respectively compared with the HFD group, which indicated that the oligopeptide could effectively improve the obesity characteristics of the NAFLD mice.

[0056] The white adipose tissue was cut into small pieces of 0.5 cm x 0.5 cm x 0.2 cm. The tissue pieces were fixed in 10% neutral formalin (pH 7.2) at 4°C for 24 hours (5 times the volume of the fixing solution to the tissue pieces to avoid lipid dissolution). Subsequently, gradient alcohol dehydration was performed: 70% alcohol: 1 hour of immersion; 80% alcohol: 1 hour of immersion; 95% alcohol I: 1 hour of immersion; 95% alcohol II: 1 hour of immersion; anhydrous alcohol I: 30 minutes of immersion; anhydrous alcohol II: 30 minutes of immersion. Then, xylene I: 20 minutes of immersion; xylene II: 20 minutes of immersion. Embedding: the tissue pieces were placed in melted paraffin (56°C), 3 times of wax immersion (30 minutes each time), and then fixed with an embedding frame, and cooled and solidified into wax blocks. Sectioning: the wax blocks were cut into 5 pm-thick sections using a paraffin sectioning machine, and attached to glass slides treated with polylysine. Unfolding: the glass slides were placed in a 37°C water bath to unfold the sections (the water temperature should not be too high to avoid melting of the fat droplets), and then placed in a 60°C oven for 2 hours to firmly attach the sections. De-waxing to water: xylene I: 10 minutes of immersion; xylene II: 10 minutes of immersion; anhydrous alcohol I: 5 minutes of immersion; anhydrous alcohol II: 5 minutes of immersion; 95% alcohol: 5 minutes of immersion; 80% alcohol: 5 minutes of immersion; 70% alcohol: 5 minutes of immersion; distilled water: 5 minutes of immersion.

[0057] Hematoxylin staining: the sections were placed in Harris hematoxylin staining solution for 5 minutes. Differentiation: differentiation was performed using 1% hydrochloric acid alcohol (70% alcohol) for 10 seconds, and tap water was used for 10 minutes of rinsing to return to blue. Eosin staining: placed in 0.5% eosin staining solution for 1 minute (cytoplasm and matrix are pink), and quickly rinsed with tap water. Dehydration: 70% alcohol: 30 seconds of immersion; 80% alcohol: 30 seconds of immersion; 95% alcohol I: 1 minute of immersion; 95% alcohol II: 1 minute of immersion; anhydrous alcohol I: 2 minutes of immersion; anhydrous alcohol II: 2 minutes of immersion. Transparency: xylene I: 3 minutes of immersion; xylene II: 3 minutes of immersion. Mounting: neutral gum (or Canada balsam) was dropped on the sections, a cover glass was added (to avoid air bubbles), and air-dried at room temperature. Finally, observation and photography were performed under an inverted microscope.

[0058] The collected blood was centrifuged at 1600g for 10 min at 4°C to obtain serum. The contents of total cholesterol (TC), triglyceride (TG), non-esterified fatty acids (NEFA), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the serum were determined by biochemical kits (Nanjing Jiancheng Bioengineering Institute, China). These detections were performed according to the instructions provided by the manufacturer of the biochemical kit.

[0059] The detection results are shown in Figure 5 Figure 5 Figure A in the middle is a white fat tissue H&E staining result graph. It can be seen that compared with the ND group, the morphology of fat cells in the HFD group mice is disordered, and the volume is significantly increased. However, under the intervention of oligopeptide D and oligopeptide E for 14 weeks, the morphology of fat cells is significantly improved and the cell diameter is significantly reduced. These results show that the intervention of oligopeptide can effectively alleviate the abnormal enlargement of fat cells induced by high-fat diet. The abnormal increase of TC, TG, and HDL-C levels in serum is one of the main characteristics of lipid metabolism disorder in NAFLD mice induced by high-fat diet. Further determination of blood lipid spectrum, the results are shown in Figure 5 Figures B, C, D, E, and F in the middle, the results show that under the intervention of long-term high-fat feed, the TC, TG, and LDL-C levels in the serum of HFD group mice are significantly increased compared with ND group mice, increased by 85.16%, 133.00%, and 179.75%, respectively, and the HDL-C level is significantly reduced (decreased by 45.70%). However, these abnormal changes in blood lipid levels were significantly reversed under the administration of oligopeptide D and oligopeptide E, and the blood lipid levels of NAFLD mice (HFD group) induced by high-fat diet returned to the ND group. It is worth noting that the administration of oligopeptide D cannot significantly reduce the LDL-C level in the serum of NAFLD mice.

[0060] ​The liver tissue of each group of mice was selected for target area (such as the left lobe of the liver) and cut into small pieces of 0.3 cm x 0.3 cm x 0.2 cm, and H&E staining and Oil Red O staining were performed, respectively. The Oil Red O experimental method: the liver tissue block was fixed with 4% paraformaldehyde for 1.5 hours (4°C, to avoid excessive fixation leading to lipid droplet destruction), then immersed in 30% sucrose solution at 4°C overnight. The tissue block was embedded in OCT embedding agent, frozen at -20°C for 30 minutes, cut into 6μm thick sections with a frozen section machine, attached to a glass slide, and air dried at room temperature for 30 minutes. The section was soaked in 60% isopropanol for 5 minutes. Oil Red O staining: immerse in freshly prepared oil red O staining solution (0.5g oil red O dissolved in 100ml isopropanol, take 60ml staining solution + 40ml distilled water dilution, filter before use), 37°C staining for 15 minutes. Differentiation: 60% isopropanol washes off excess dye for 1 minute, tap water rinse. Nucleus counterstaining: hematoxylin staining for 30 seconds, tap water rinse to blue. Mounting: use glycerol gelatin mounting. Finally, observe and take pictures under an inverted microscope.

[0061] The liver tissue of each group of mice was selected for target area (such as the left lobe of the liver) and cut into small pieces of 0.3 cm x 0.3 cm x 0.2 cm, and H&E staining and Oil Red O staining were performed, respectively. The Oil Red O experimental method: the liver tissue block was fixed with 4% paraformaldehyde for 1.5 hours (4°C, to avoid excessive fixation leading to lipid droplet destruction), then immersed in 30% sucrose solution at 4°C overnight. The tissue block was embedded in OCT embedding agent, frozen at -20°C for 30 minutes, cut into 6μm thick sections with a frozen section machine, attached to a glass slide, and air dried at room temperature for 30 minutes. The section was soaked in 60% isopropanol for 5 minutes. Oil Red O staining: immerse in freshly prepared oil red O staining solution (0.5g oil red O dissolved in 100ml isopropanol, take 60ml staining solution + 40ml distilled water dilution, filter before use), 37°C staining for 15 minutes. Differentiation: 60% isopropanol washes off excess dye for 1 minute, tap water rinse. Nucleus counterstaining: hematoxylin staining for 30 seconds, tap water rinse to blue. Mounting: use glycerol gelatin mounting. Finally, observe and take pictures under an inverted microscope.

[0062] The liver tissue of each group of mice was selected for target area (such as the left lobe of the liver) and cut into small pieces of 0.3 cm x 0.3 cm x 0.2 cm, and H&E staining and Oil Red O staining were performed, respectively. The Oil Red O experimental method: the liver tissue block was fixed with 4% paraformaldehyde for 1.5 hours (4°C, to avoid excessive fixation leading to lipid droplet destruction), then immersed in 30% sucrose solution at 4°C overnight. The tissue block was embedded in OCT embedding agent, frozen at -20°C for 30 minutes, cut into 6μm thick sections with a frozen section machine, attached to a glass slide, and air dried at room temperature for 30 minutes. The section was soaked in 60% isopropanol for 5 minutes. Oil Red O staining: immerse in freshly prepared oil red O staining solution (0.5g oil red O dissolved in 100ml isopropanol, take 60ml staining solution + 40ml distilled water dilution, filter before use), 37°C staining for 15 minutes. Differentiation: 60% isopropanol washes off excess dye for 1 minute, tap water rinse. Nucleus counterstaining: hematoxylin staining for 30 seconds, tap water rinse to blue. Mounting: use glycerol gelatin mounting. Finally, observe and take pictures under an inverted microscope.

[0063] The detection results are shown in Table 1, and the liver tissue staining results are shown in Figure 1. Figure 6 ​Figure 6 As shown in FIG. 2A, a large number of lipid vacuoles were observed in the liver of long-term high-fat diet-induced NAFLD mice, accompanied by focal necrosis and a small amount of inflammatory infiltration, showing severe liver damage. After the intervention of two different doses of oligopeptides, the number and size of lipid vacuoles in the liver showed a dose-dependent reduction, while the intercellular space was reduced and there was no obvious cell infiltration, and the pathological characteristics were significantly improved. Oil red O staining further showed that the liver cells of the ND group were regular in shape, with clear boundaries between cells, no lipid accumulation and degeneration; while the cells of the HFD group were arranged in disorder, the structure and edge of the cells were blurred, and were accompanied by a large number of fat droplets and lipid accumulation, showing severe liver fat infiltration and fatty degeneration. After 14 weeks of oligopeptide administration, the fat deposition induced by high-fat diet was improved, and the liver damage was significantly recovered. As shown in FIG. 2B, the content of TC in the liver tissue of the HFD group was 53.99% higher than that of the ND group. As shown in FIG. 2C, the content of TG in the liver tissue of the HFD group was 82.71% higher than that of the ND group. As shown in FIG. 2D, the content of LDL-C in the liver tissue of the HFD group was 81.84% higher than that of the ND group. As shown in FIG. 2E, the content of HDL-C in the liver tissue of the HFD group was significantly lower than that of the ND group. The content of HDL-C in the liver tissue of the D group and the E group was 33.80% and 37.97% higher than that of the HFD group, respectively. Figure 6 As shown in FIG. 2B, the content of TC in the liver tissue of the HFD group was 53.99% higher than that of the ND group. As shown in FIG. 2C, the content of TG in the liver tissue of the HFD group was 82.71% higher than that of the ND group. As shown in FIG. 2D, the content of LDL-C in the liver tissue of the HFD group was 81.84% higher than that of the ND group. As shown in FIG. 2E, the content of HDL-C in the liver tissue of the HFD group was significantly lower than that of the ND group. The content of HDL-C in the liver tissue of the D group and the E group was 33.80% and 37.97% higher than that of the HFD group, respectively. Figure 6 As shown in FIG. 2F and FIG. 2G, compared with the ND group, the ALT and AST activities of the HFD group increased significantly. The ALT activity increased from 30.37 U / L to 60.62 U / L, and the AST activity increased from 48.70 U / L to 117.14 U / L. The intervention of low-dose and high-dose oligopeptides significantly reversed this trend. The ALT activity of the D group and the E group was 21.88% and 36.85% lower than that of the HFD group, respectively, and the AST activity was 35.36% and 41.79% lower than that of the HFD group, respectively. The above results fully illustrate that the intervention of oligopeptides can significantly alleviate the liver damage of NAFLD mice induced by high-fat diet.

[0064] The collected mouse liver tissues were completely homogenized in a homogenizer, and then centrifuged at 8000g at 4°C for 10 min to obtain the supernatant. The contents of malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH) in the supernatant were determined by biochemical kits (Nanjing Jiancheng Bioengineering Institute, China). These detections were performed according to the instructions provided by the manufacturer of the biochemical kit.

[0065] Results are shown in Figure 7 Figures A, B and C, respectively. Figure 7 Figures A, B and C, respectively. MDA reflects the severity of free radical damage to liver cells. The results show that the MDA level in the liver of the HFD group mice was significantly higher than that in the ND group, while the upward trend of MDA level in the D and E groups of mice was significantly inhibited, accounting for only 62.46% and 57.76% of the HFD group (as shown in Figure 7 Figure D). The above results show that the oligopeptide can effectively reduce the oxidative stress level of the liver of the NAFLD mice.

[0066] Example 4: Investigation of the improvement effect of D peptide and E peptide on insulin resistance, the method is as follows:

[0067] (1) After 14 weeks of oligopeptide intervention, C57BL / 6 mice were fasted for 12 hours overnight (free water), and then blood was taken from the tail vein (cut off 1mm of the tail tip, 5μL of blood was sucked with a capillary), and then a portable blood glucose meter (Roche vitality type) was used to directly detect the whole blood glucose;

[0068] (2) The separated serum was collected and detected by ELISA kit (Mercodia mouse insulin kit) to calculate the concentration of mouse serum insulin;

[0069] (3) After 14 weeks of oligopeptide intervention, C57BL / 6 mice were weighed after fasting for 12 hours overnight, and then 20% glucose solution was administered at 2g / kg body weight. Before administration (0 minutes, i.e. fasting blood glucose), 30 minutes, 60 minutes, 120 minutes and 180 minutes after administration, blood was collected according to the "fasting blood glucose" method to detect the blood glucose concentration;

[0070] (4) Area under the curve (AUC): usually used to quantify the overall blood glucose level of OGTT curve, reflecting the total blood glucose exposure after glucose load, which can better reflect glucose intolerance than single time point. AUC calculation method: based on the blood glucose value (C0, C30, C60, C120, C180, unit: mmol / L) at each time point of OGTT, the time interval is in minutes (Δt): Unit: mmol L - ¹ min.

[0071] The QUICK index is an insulin sensitivity evaluation index based on fasting blood glucose and insulin. The higher the index, the better the insulin sensitivity. The calculation method is as follows: fasting blood glucose (FBG) and fasting insulin (FINS) are required, and the formula is:

[0072] The HOMA-IR index is the most commonly used insulin resistance evaluation index, which is based on fasting blood glucose and insulin. The higher the index, the more severe the insulin resistance. The calculation method is as follows: fasting blood glucose (FBG, mmol / L) and fasting insulin (FINS, mU / L) are required, and the formula is: .

[0073] As shown in FIGS. A and B of Figure 8 , compared with the ND group, the serum insulin level of the HFD group mice was significantly increased. Compared with the HFD group mice, the serum insulin level of the high-fat diet group mice (D and E groups) treated with different oligopeptides was significantly decreased. The HOMA-IR (homeostatic model assessment of insulin resistance index) was further calculated from the FBG and serum insulin level.

[0074] As shown in FIGS. C, D and E of Figure 8 , in the OGTT experiment, after intragastric administration of glucose solution, the blood glucose level of each group increased sharply, and the highest value reached was positively correlated with the relative level of FBG of each group. The highest blood glucose level of the HFD group was 23.16 mmol / L, which was 156.06% of the ND group (14.84 mmol / L). The highest blood glucose level of the D group and the E group treated with oligopeptides for 14 weeks was only 86.83% and 78.84% of the HFD group. Among them, the blood glucose level of the HFD group mice reached the peak at 30 min and then decreased, while the blood glucose level of the ND group, the OP group, the D group and the E group mice showed an earlier downward trend. The blood glucose level of the four groups reached the peak at 15 min and showed a downward trend at 30 min. At the end of the 120 min OGTT experiment, the blood glucose level of the ND group, the OP group, the D group and the E group was significantly lower than that of the HFD group, which was only 83.03%, 74.46%, 54.96% and 50.45% of the HFD group. Further quantification of the area under the OGTT curve showed that compared with the HFD group, the area under the OGTT curve of the D group and the E group within 120 min was reduced by 18.84% and 32.88%, respectively, showing a dose-dependent decrease. This result shows that oligopeptides can significantly improve the glucose tolerance of NAFLD mice.

[0075] As shown in FIGS. A and B of Figure 8As shown in FIG. 5, the HOMA-IR index of the HFD group mice was significantly increased, which was increased by 70.52% compared with the ND group, while this increasing trend was significantly inhibited by the oligopeptide intervention of the D group and the E group. Compared with the HFD group, the HOMA-IR index of the D group and the E group was decreased by 30.20% and 49.12%, respectively. The above results fully show that the oligopeptide can significantly improve the insulin resistance of the NAFLD mice induced by high-fat diet.

[0076] Example 5: In order to further explore the regulation mechanism of the D peptide and the E peptide on the abnormal lipid metabolism of the NAFLD mice, the expression levels of the related proteins in the lipid metabolism pathway were determined by Western blotting, and the method was as follows:

[0077] The liver tissue sample was rinsed with 1xPBS pre-cooled in an ice bath for three times to remove blood residues, and then was transferred to a homogenizing tube after being cut into pieces. RIPA buffer containing 1% protease inhibitor was added at 10 times the volume of the tissue, and two 2-mm magnetic beads were added for homogenization treatment at 60 seconds. The homogenized sample tube was taken out of the ice bath for half an hour, and was vortexed for 30 seconds every 5 minutes to promote the complete lysis of the tissue. The mixture was centrifuged at 12000 g at 4°C for 10 minutes, and the supernatant was collected as the total protein component. The protein content in the supernatant was quantified using a commercial BCA protein detection kit. Subsequently, the extracted protein sample was mixed with 5xprotein loading buffer at a ratio of 4:1 (v / v), and was denatured at 100°C for 15 minutes. The concentrated gel voltage was set to 75 V, and the separation gel voltage was set to 120 V. The membrane was transferred at 300 milliampere for 30 minutes. Then, the blocking was performed at room temperature in 5% skimmed milk powder containing 0.5% TBST for 1 hour. The primary antibody of the phosphorylated protein was diluted with 5% skimmed milk powder and 5% BSA dissolved in TBST, and was incubated at 4°C overnight. The secondary antibody was diluted with TBST at 1:3000, and was incubated for 5 minutes each time on a destaining shaker at room temperature. After incubation at room temperature for 30 minutes, the membrane was washed with TBST for three times, each for 5 minutes. Western blotting detection was performed using an ECL chemiluminescence kit (Thermo Fisher Scientific, California, USA), and the band optical density was analyzed by Image J software.

[0078] The results are shown in FIGS. 6 and 7. Figure 9 and Figure 10 Figure 9 ​Results showed that: in normal diet (ND) and control treatment (OP) groups, the AdipoR2-AMPK-AKT pathway was continuously activated (p-AdipoR2, p-AMPK, p-AKT ratio was higher), the expression of glucose transporter GLUT4 was sufficient, glycogen synthase kinase GSK3β remained active (p-GSK3β ratio was low), and the transcription factor FoxO1 was in dephosphorylated state (p-FoxO1 ratio was low); high-fat diet (HFD) significantly inhibited the pathway, leading to decreased expression of GLUT4 (glucose uptake was hindered), increased phosphorylation of GSK3β (glycogen synthesis was inhibited), and abnormal up-regulation of FoxO1 phosphorylation (glucolysis regulation disorder); intervention treatment (D peptide, E peptide) can effectively restore the activity of the pathway, promote the expression of GLUT4, GSK3β activation and FoxO1 phosphorylation state reset, and the difference is statistically significant. In summary, HFD disturbs glucose metabolism by blocking the AdipoR2-AMPK-AKT signaling axis, while D peptide and E peptide intervention can restore the pathway and improve glucose uptake, glycogen synthesis and gluconeogenesis balance. Figure 10 It was shown that long-term high-fat diet induced a significant decrease in AMPK phosphorylation, while oligopeptide intervention significantly activated AMPK phosphorylation expression. The expression levels of D and E groups were up-regulated by 43.71% and 57.47% compared with the HFD group. At the same time, the intervention of oligopeptide significantly up-regulated the expression of PPAR-α and CPT-1. Compared with the HFD group, the low-dose and high-dose oligopeptide intervention up-regulated the expression of PPAR-α by 40.23% and 58.35%, respectively; and up-regulated the expression of CPT-1 by 54.75% and 71.36%, respectively. As the main regulator of liver lipid synthesis genes, the expression level of SREBP-1 in the liver of NAFLD mice induced by high-fat diet was significantly increased, and the expression level of HFD group was increased by 48.91% compared with ND group. After giving high-dose oligopeptide intervention, the expression of SREBP-1 was reduced by 38.46%, while the low-dose oligopeptide had no obvious inhibitory effect on the abnormal increase of SREBP-1 expression induced by high-fat diet. The above results show that oligopeptides D and E can activate the AMPK / PPAR-α pathway by up-regulating the expression of AMPK phosphorylation, thereby up-regulating the expression of CPT1 and further enhancing the level of fatty acid oxidation; at the same time, by down-regulating the expression of SREBP-1, it reduces the lipid deposition caused by high-fat diet.

[0079] Example 6: Spatial structure matching, chemical group recognition and hydrogen bonding interaction The molecular docking simulation method was used, as follows:

[0080] The structure of the control peptide KRQKYD and the target test peptide (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) was predicted by AlphaFold, the structure of the AdipoR2 receptor protein was directly obtained from the PDB database, the docking region (surrounding the target binding site) was set, the genetic algorithm was used to search for the best binding conformation of the polypeptide and the target. According to the spatial complementarity (contact area, conformation fitting degree, RMSD value) of the conformation, the score (0-10 points, the higher the fitting degree, the higher the score) was self-defined. 8-10 points: the conformation is almost completely matched, no spatial conflict; 0-5 points: the conformation is distorted, and the spatial conflict is serious. According to the chemical group recognition (binding enthalpy change, binding constant and stoichiometric ratio) of the single binding site model, the score (0-10 points, the higher the functional group matching degree and interaction strength, the higher the score) was self-defined. 8-10 points: the functional groups are completely complementary, strong interaction (π-π stacking, electrostatic synergy); 0-5 points: functional group conflict, weak interaction. The results are shown in Table 2:

[0081] Table 2 Analysis results of the binding ability of target oligopeptide to AdipoR2 receptor

[0082]

[0083] The interaction scores between the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) in Table 2 are all >5 points, indicating that the intermolecular interaction force between the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) and the AdipoR2 receptor protein is stronger than that of the control peptide KRQKYD. At the same time, the number of hydrogen bonds formed between the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) and the AdipoR2 receptor protein is significantly more than that of the control peptide KRQKYD, indicating that the hydrogen bond binding ability of the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, YHEHRSDLN) to the AdipoR2 receptor protein is stronger than that of the control peptide KRQKYD.

[0084] The total score is obtained by adding the spatial structure matching score (0-10), the chemical group identification score (0-10), and the number of hydrogen bonds (1). The higher the score, the stronger the binding ability of the oligopeptide to the AdipoR2 receptor protein. The total scores of the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, and YHEHRSDLN) were significantly higher than that of KRQKYD, indicating that the target test peptides (YKATEPVIAF, INKVEELKKKY, GEKLKRQKY, ELIDQDARDLY, and YHEHRSDLN) had stronger specific binding ability to the AdipoR2 receptor protein than KRQKYD.

[0085] Example 7: The D peptide and the E peptide in Example 1 are prepared into soft candies as follows:

[0086] (1) The D peptide and the E peptide (molar ratio 1:1) are dissolved in water to obtain a polypeptide aqueous solution at a final concentration of 200 mg / mL;

[0087] (2) The fat part of the three-year Jinhua ham is mixed with petroleum ether at a solid-liquid ratio of 1 g:5 mL, and then ultrasonic extraction is performed at 50°C for 2 h. The supernatant is taken, and the petroleum ether is removed by rotary evaporation to obtain ham fat;

[0088] (3) The polypeptide aqueous solution, the ham fat, and the castor glycerol oleate are mixed and stirred at a volume ratio of 60:40:3.2, and then high-speed shearing is performed at 25000 rpm (40 s / time, 4 times) to obtain an emulsion with the function of improving sugar and lipid metabolism disorder for standby use.

[0089] (4) 60 parts of the above emulsion, 1 part of vitamin C, 3 parts of apple concentrate, 0.5 parts of malt dextrin, 0.5 parts of xylitol, 1 part of carrageenan, and 1 part of xanthan gum are mixed and heated at 70°C for 25 min. The mixture is then injected into a mold and cooled to form a soft candy. The apple concentrate can be replaced by concentrated fruit juice of banana, strawberry, orange, watermelon, etc.

[0090] Example 7: The D peptide or the E peptide in Example 1 is prepared into a soft capsule as follows:

[0091] (1) The D peptide or the E peptide is dissolved in water to obtain a polypeptide aqueous solution at a final concentration of 100 mg / mL;

[0092] (2) The fat part of the three-year Jinhua ham is mixed with petroleum ether at a solid-liquid ratio of 1.5 g:7 mL, and then ultrasonic extraction is performed at 45°C for 4 h. The supernatant is taken, and the petroleum ether is removed by rotary evaporation to obtain ham fat;

[0093] (3) mixing the polypeptide aqueous solution with ham fat and castor glycerol oleate by volume ratio of 70:30:1.8, stirring, and high-speed shearing at 22000 rpm (40 s / time, 4 times) to obtain an emulsion for improving glycolipid metabolism disorder.

[0094] (4) mixing 70 parts of the emulsion with 2 parts of vitamin C to obtain a capsule content; using gelatin: water: glycerol = 80-100:80-100:20-40 as raw materials to prepare a soft capsule shell, and encapsulating the capsule content into the soft capsule shell to obtain a soft capsule.

Claims

1. A dry-cured ham-derived oligopeptide, characterized in that, The amino acid sequence of the oligopeptide is YHEHRSDLN.

2. The use of the oligopeptide from dry-cured ham in the preparation of a drug for treating at least one of hyperglycemia, diabetes, hyperlipidemia and fatty liver.

3. Use according to claim 2, characterized in that, The content of the oligopeptide from dry-cured ham in the drug is 1-200 mg / mL.

4. The use of an oligopeptide with an amino acid sequence of ELIDQDARDLY in the preparation of a drug for treating at least one of hyperglycemia, diabetes, hyperlipidemia and fatty liver.

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