A tetrapeptide FP4 with the function of alleviating diabetic kidney damage and its preparation method and application

Abstract

The present invention discloses a tetrapeptide FP4 that can alleviate diabetic kidney damage, as well as its preparation method and application, belonging to the field of biotechnology. The tetrapeptide FP4, obtained by screening from Pacific salmon sperm protease hydrolysate, has an amino acid sequence of FGVP and can significantly reduce fasting blood glucose, serum creatinine, urea nitrogen, and urine protein in diabetic mice. It has superior activity and fewer toxic side effects than the positive drug metformin, and can be used in functional foods that alleviate diabetic kidney damage.

Description

Technical Field

[0001] The present invention relates to a small molecule peptide and a preparation method and application thereof, and specifically to a tetrapeptide FP4 capable of alleviating diabetic kidney damage and a preparation method thereof and application thereof in preparing a drug for alleviating diabetic kidney damage, belonging to the field of biotechnology. Background Art

[0002] Diabetes mellitus has become a global metabolic disease threatening human health, with its incidence showing a significant upward trend. Type 2 diabetes accounts for approximately 95% of diabetes types, characterized by decreased insulin receptor sensitivity, pancreatic β-cell dysfunction, and dysregulated hepatic gluconeogenesis. Recent studies have confirmed that rapid degradation of glucagon-like peptide-1 (GLP-1) is a key mechanism for impaired glucose metabolism, and that dipeptidyl peptidase-IV (DPP-IV) is a key enzyme that catalyzes GLP-1 inactivation. Therefore, specific inhibition of DPP-IV activity has become an important research focus for ameliorating insulin secretion defects in type 2 diabetes.

[0003] As a serine protease with a molecular weight of approximately 110 kDa, DPP-IV holds significant clinical value in the treatment of diabetes. Although chemical inhibitors such as vildagliptin and alogliptin are widely used, they may induce adverse reactions such as joint pain and pancreatitis. Against this backdrop, screening for highly effective and low-toxic DPP-IV inhibitors from natural products has become a research hotspot. Among these, active peptides obtained from food-derived proteolysis have shown unique potential for modulating intestinal microbiota and improving insulin resistance due to their excellent biocompatibility, low metabolic burden, and high safety threshold.

[0004] Specific enzymatic hydrolysis of proteins from different sources can yield a diverse array of active peptides with diverse structures and functions. DPP-IV inhibitory peptides do not exhibit a specific amino acid motif; their activity is closely related to physicochemical properties such as spatial conformation and hydrophobicity. Notably, even with the same protein source, different enzymatic hydrolysis processes (e.g., enzyme type, temperature, and pH) can result in significant differences in product composition. This provides a theoretical basis for the multidimensional screening of highly effective DPP-IV inhibitory peptides.

[0005] Recently, a number of studies have revealed the functions of active peptides from different sources against diabetes and its complications. Jiang Meiling et al. found that the half-inhibitory concentration (IC50) of oyster hydrolysate for DPP-IV was 2.60±0.46 mg / mL (Structural Characterization of Oyster Hypoglycemic Peptides and Their Synergistic Effect with Anthocyanins, Jiang Meiling, Chen Zhongqin, Qin Xiaoming et al., Journal of Dalian Ocean University, 2023, 38(03): 455-463). Yiyun Zhang et al. identified the heptapeptide GPVRGPF and the hexapeptide HPHPHL from bovine milk protein hydrolysate with good DPP-IV inhibitory activity (Mining Bovine Milk Proteins for DPP-4 Inhibitory Peptides Using Machine Learning and Virtual Proteolysis, Zhang Y, Zhu Y, Bao X, Dai Z, Shen Q, Wang L, Xue Y, Research (Wash DC), 2024 Jun 17;7: 0391). Ren Guoyan's team screened out a new antidiabetic peptide (hexapeptide GPAGAP) from Andrias davidianus collagen hydrolysates (Anovel antidiabetic peptide GPAGAP from Andrias davidianus collagen hydrolysates: screening, action mechanism prediction and improving insulin resistance in HepG2 cells, Dong ZH, Pan RY, Ren GY, et al, Food & Medicine Homology, 2024, 1(1): 9420010). Ning An et al. found that after buffalo casein was hydrolyzed by a compound flavor protease for 3 hours, the DPP-IV half-inhibitory concentration (IC50) value of the hydrolyzate was 1.04 mg / mL, and further identified the nonapeptide YPFPGPIPN, whose IC50 value was 0.88 mg / mL (Enzymatic hydrolysis of buffalo casein enhances DPP-4inhibition: Structural modifications and bioactive peptide identification, AnN, Yang J, Zhang Y, Suo H, Song J, J Dairy Sci, 2025 Mar, 108 (3): 2169-2181).Ma Xinyue et al. summarized hypoglycemic peptides from various sources with clear structural characteristics (Research Progress of Animal-derived Hypoglycemic Peptides, Ma Xinyue, Liu Mingyu, Li Rong et al., Food Industry Science and Technology, 2022, 43(22): 438−444). The sequence lengths of these hypoglycemic active peptides are concentrated in the range of 2-8 amino acids, and proline and alanine appear frequently in the active sites.

[0006] The above reports do not mention peptides with the same or similar structures as the active peptides of the present invention that can alleviate diabetic kidney damage. In addition, if modifications are to be made based on the reported active peptide sequences, the amount of data will increase exponentially, making it impractical, as there are 20 common amino acids to choose from at each amino acid position.

[0007] The salmon belongs to the order Salmoniformes, family Salmonidae, and genus Salmon in biological taxonomy. It is also called Pacific salmon in my country. Its scientific name is Oncorhynchus keta Protamine is a migratory pelagic fish that is high in protein, low in fat, and rich in ω-3 polyunsaturated fatty acids. It is an important aquatic resource with both nutritional and ecological value. Protamine, rich in arginine and histidine, is a high-quality raw material for the preparation of antimicrobial peptides, immunomodulatory peptides, and metabolically active peptides. Summary of the Invention

[0008] The purpose of the present invention is to provide a small molecule peptide screened from Pacific salmon sperm protein hydrolysate, which has the function of alleviating diabetic kidney damage and can be used in drugs for alleviating diabetic kidney damage.

[0009] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0010] A tetrapeptide FP4 having the function of alleviating diabetic kidney damage, wherein the amino acid sequence of the tetrapeptide FP4 is FGVP, as shown in SEQ ID NO: 2 in the sequence table, and has the function of alleviating diabetic kidney damage.

[0011] The use of the aforementioned tetrapeptide FP4 having the function of alleviating diabetic kidney damage in the preparation of a drug for alleviating diabetic kidney damage.

[0012] The preparation method of the aforementioned tetrapeptide FP4 having the function of alleviating diabetic kidney damage adopts a solid phase synthesis method, specifically:

[0013] The solid phase synthesis was carried out using Fmoc-protected amino acids as raw materials and polystyrene resin as a solid phase carrier using the Fmoc solid phase synthesis strategy.

[0014] The preparation method of the aforementioned tetrapeptide FP4 having the function of alleviating diabetic kidney damage adopts an enzymatic hydrolysis method, specifically:

[0015] (1) Take Pacific salmon sperm, remove impurities, add water, and heat to 65±2℃ for 12 hours;

[0016] (2) Remove the fish essence and beat it into a pulp, put it into a reactor, add water, start the stirrer and heat it to 48°C, add papain for enzymatic hydrolysis for 2 hours, continue to heat it to 57°C, add nuclease, deaminase, alkaline protease and neutral protease for enzymatic hydrolysis for 3 hours, then add flavor protease for enzymatic hydrolysis for 4 hours, continue to heat it to 85°C and maintain it for 30 minutes to obtain the enzymatic hydrolyzate;

[0017] (3) Adding the chitosan aqueous solution to the enzymatic hydrolysate while cooling, centrifuging after floccules appear, retaining the supernatant and filtering to obtain a clear and transparent filtrate;

[0018] (4) Adjust the pH value of the filtrate to 5.50±1.50, then add coconut shell activated carbon, decolorize and deodorize at 70±2℃ for 30min, filter again, desalinate and concentrate the filtrate to obtain a concentrate;

[0019] (5) The concentrated liquid is dried to obtain Pacific salmon protamine polypeptide, which contains a large amount of tetrapeptide FP4.

[0020] The present invention is beneficial in that:

[0021] (1) The tetrapeptide FP4 obtained from the proteolytic solution of Pacific salmon sperm has the function of alleviating diabetic kidney damage and can significantly reduce the fasting blood glucose, serum creatinine, urea nitrogen and urine protein levels in diabetic mice. It has better activity than the positive drug metformin and can be used in drugs to alleviate diabetic kidney damage.

[0022] (2) The tetrapeptide FP4 provided by the present invention is obtained by screening the protease hydrolysate of Pacific salmon sperm. Compared with metformin, it has the advantages of less toxic side effects and higher safety, and can be taken for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 3D schematic diagram of the binding mode of tetrapeptide FP4 and DPP-IV;

[0024] Figure 2 2D schematic diagram of the binding mode of tetrapeptide FP4 and DPP-IV;

[0025] Figure 3 This is a graph showing the effect of tetrapeptide FP4 on the body weight of diabetic mice, *** indicates P < 0.001;

[0026] Figure 4 This is the effect of tetrapeptide FP4 on fasting blood glucose in diabetic mice, *** indicates P < 0.001;

[0027] Figure 5 This is the effect of tetrapeptide FP4 on urinary protein in diabetic mice, ** indicates P < 0.01, *** indicates P < 0.001;

[0028] Figure 6 This is the effect of tetrapeptide FP4 on serum creatinine in diabetic mice, *** indicates P < 0.001;

[0029] Figure 7 This is a graph showing the effect of tetrapeptide FP4 on serum urea nitrogen in diabetic mice. *** indicates P < 0.001. DETAILED DESCRIPTION

[0030] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] 1. Preparation of Pacific salmon protamine polypeptide

[0032] Take Pacific salmon sperm, remove impurities, add appropriate amount of purified water, heat to 65±2℃, and maintain for 12 hours (to allow the testicular tissue to fully swell, facilitating extraction and subsequent impurity removal), and stir every 4 hours.

[0033] The fish essence was removed and slurried twice with a bone mud machine, then placed in a reactor. Purified water was added at a solid-liquid ratio of 1:5. The stirrer was started (speed 100 rpm) and the temperature was raised to 48°C. Papain (enzyme substrate ratio 1.0%) was added for enzymatic hydrolysis for 2 h. The temperature was continued to be raised to 57°C. Nuclease (enzyme substrate ratio 0.5%), deaminase (enzyme substrate ratio 0.3%), alkaline protease (Alcalase® 2.4 L, enzyme substrate ratio 1.0%), and neutral protease (Neutrase® 0.8 L, enzyme substrate ratio 0.5%) were added for enzymatic hydrolysis for 3 h. Subsequently, flavor protease (Flavourzyme® 500 MG, enzyme substrate ratio 1.0%) was added for enzymatic hydrolysis for 4 h. The temperature was continued to be raised to 85°C and maintained for 30 min (enzyme inactivation) to obtain the enzymatic hydrolyzate.

[0034] While cooling, a 1 wt% chitosan aqueous solution (food grade, to promote the sedimentation of impurities) was added to the enzymatic hydrolyzate. After floccules appeared, the solution was centrifuged (5000 rpm, 3 min) to retain the supernatant, which was filtered using a plate and frame filter press at a pressure of 0.3 MPa to obtain a clear and transparent filtrate.

[0035] The filtrate was placed in a reactor, and the pH value of the filtrate was adjusted to 5.50±1.50. 1% of the total volume of the liquid was then added with coconut shell activated carbon. The mixture was decolorized and deodorized at 70±2°C for 30 minutes. The mixture was filtered again using a plate and frame filter press. The obtained filtrate was desalted using a nanofiltration system. When salt was no longer detected in the discharged liquid, the mixture was transferred to a double-effect concentrator for further concentration to obtain a concentrated liquid.

[0036] The concentrate was spray dried using a spray dryer with a feed pressure of 18.0 MPa, a flow rate of 300 L / h, an air inlet temperature controlled at 130°C, and an air outlet temperature controlled at 95°C to obtain Pacific salmon protamine polypeptide (beige powder).

[0037] 2. Obtaining the Sequence of the Pacific Salmon Protamine Polypeptide

[0038] The Pacific salmon protamine polypeptide obtained above was detected by LC-MS / MS, and the detection results were analyzed by mass spectrometry analysis software to obtain several polypeptide sequences.

[0039] LC-MS / MS detection conditions are:

[0040] (1) In the liquid phase method: the chromatographic column is C18, 3 μm, 250 mm × 75 μm (Eksigent), phase A is water, 0.1% formic acid; phase B is acetonitrile, 0.1% formic acid, the flow rate is 300 nL / min, the injection volume is 1 μL, and the chromatographic gradient is 70 min. The specific elution gradient is: 0-55 min, phase A is uniformly reduced from 95% to 65%; 55-63 min, phase A is uniformly reduced from 65% to 50%; 63-64 min, phase A is uniformly reduced from 50% to 0; 64-70 min, maintain 0% phase A;

[0041] (2) Mass spectrometry: Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific), positive ion detection mode, primary resolution 120,000, AGC setting 310, scan range 110–2000 m / z. MIPS mode for peptides, valence states 1–6, secondary resolution 17,500, separation window 1.6 m / z.

[0042] 3. Screening peak area ≥10 7 Active oligopeptides with amino acid number ≤ 6

[0043] From the peptide sequences obtained above, 11 peptide sequences with peak areas ≥10 7 The screening results of active oligopeptides with amino acid number ≤ 6 are shown in Table 1.

[0044] Table 1 Peak area ≥10 in Pacific salmon protamine peptides 7 Active oligopeptide sequence with amino acid number ≤ 6

[0045]

[0046] 4. Screening of active oligopeptides with strong binding ability to DPP-IV

[0047] The 11 active oligopeptides in Table 1 were docked with the dipeptidyl peptidase-4 (DPP-IV) crystal structure (PDB ID: 5Y7H) using Discovery Studio software. Before docking, the 2D structures of the active oligopeptides were converted into 3D structures by energy minimization, and active oligopeptides with strong binding ability to DPP-IV were screened.

[0048] The docking results are expressed as docking scores (-CiE). The larger the -CiE value, the stronger the interaction between the active oligopeptide and DPP-IV, and the more likely it is to alleviate diabetic kidney damage.

[0049] The molecular docking results of 11 active oligopeptides with DPP-IV are shown in Table 2.

[0050] Table 2 Prediction results of the interaction between 11 active oligopeptides and DPP-IV

[0051]

[0052] From Tables 1 and 2, we can see that among the 11 active oligopeptides, only FPVG and FGVP have peak areas exceeding 20×10 7 Among them, the -CiE of FGVP with DPP-IV is 70.7065 kcal / mol, while the -CiE of FPVG with DPP-IV is only 48.2409 kcal / mol. Therefore, FGVP (denoted as tetrapeptide FP4) was selected for further prediction analysis.

[0053] After analysis, the binding mode of tetrapeptide FP4 and DPP-IV is as follows Figure 1 and Figure 2 The molecular docking situation is as follows:

[0054] There are 10 HH bond interactions, 4 CH bond interactions, and 2 electrostatic interactions between the tetrapeptide FP4 and DPP-IV. There are 13 amino acid residues involved in the interaction between the tetrapeptide FP4 and DPP-IV, and the van der Waals force between the two is -12.13 kcal / mol.

[0055] 5. Evaluation of the Tetrapeptide FP4 in Alleviating Diabetic Kidney Damage

[0056] 1. Solid phase synthesis of tetrapeptide FP4

[0057] The tetrapeptide FP4 (amino acid sequence: FGVP) was synthesized on solid phase using the Fmoc solid phase synthesis strategy with Fmoc-protected amino acids as raw materials and polystyrene resin as the solid phase support. The purity was >90%.

[0058] 2. Animal Experiment Process

[0059] After one week of adaptive feeding, 4-6 week-old male C57BL / 6J mice were randomly divided into four groups: control group, model group, positive drug group and tetrapeptide FP4 group.

[0060] The control group was fed a standard diet, while the other groups were fed a high-sugar, high-fat diet for 4 weeks. Starting from week 5, after an 8-hour daily fast, the control group received an intraperitoneal injection of 200 μL of citric acid-sodium citrate buffer, while the other groups received an intraperitoneal injection of streptozotocin (STZ, 50 mg / kg) solution for 5 consecutive days. On day 4 of week 5, fasting blood glucose was measured in each group. A diabetic mouse model was considered successful if the fasting blood glucose level was ≥16.7 mM. Water was freely available during the experiment. After modeling, the mice were weighed and their body weights were recorded (W0).

[0061] For mice with successful modeling, the positive drug group was gavaged with 200 mg / kg metformin daily, the tetrapeptide FP4 group was gavaged with 100 mg / kg solid-phase synthesized tetrapeptide FP4 (purity>90%) daily, and the control and model groups were gavaged with normal saline (200 μL) daily. The control group was fed with ordinary feed, while the other groups continued to be fed with high-sugar and high-fat feed. They had free access to water during the experiment, which lasted for 4 weeks. At the end of the experiment, the mice were sacrificed, and their weights were weighed and recorded (W t ).

[0062] 3. Sample collection and processing

[0063] Plasma: After the last administration, mice were fasted for 12 hours, and blood was collected from the orbital venous plexus to collect 24-hour plasma. The fasting blood glucose of mice was measured using a blood glucose meter.

[0064] Urine: After the last administration, mice were fasted for 12 hours, and their 24-hour urine was collected. The supernatant was obtained by centrifugation and the 24-hour urine protein content in the urine was detected using a kit.

[0065] Serum: Collect all blood from mice by eyeball sampling, place in a 4°C refrigerator for 3 hours, and then centrifuge at 2000 rpm for 15 minutes. Aspirate the upper serum with a pipette, aliquot, and store in a -80°C refrigerator for the determination of urea nitrogen and creatinine levels in serum.

[0066] The weight gain of mice in each group (W t -W0) statistical results are shown in Figure 3 .Depend on Figure 3It can be seen that compared with the weight gain of the control group (9.98±0.88g), the weight gain of the model group (3.32±1.07g) was significantly reduced (p<0.001); compared with the weight gain of the model group (3.32±1.07g), the weight gain of the positive drug group (5.5±0.6g) was significantly increased (p<0.001), and the weight gain of the tetrapeptide FP4 group (7.71±0.55g) was also significantly increased (p<0.001), which was about 1.32 times higher than that of the model group.

[0067] The statistical results of fasting blood glucose concentration of each group of mice are shown in Figure 4 .Depend on Figure 4 It can be seen that compared with the fasting blood glucose concentration of the control group (5.45±0.3mM), the fasting blood glucose concentration of the model group (25.3±1.3mM) was significantly increased (p<0.001); compared with the fasting blood glucose concentration of the model group (25.3±1.3mM), the fasting blood glucose concentration of the positive drug group (14.5±0.7mM) was significantly decreased (p<0.001), and the fasting blood glucose concentration of the tetrapeptide FP4 group (15.2±0.63mM) was also significantly decreased (p<0.001).

[0068] The statistical results of urine protein content in each group of mice are shown in Figure 5 .Depend on Figure 5 It can be seen that compared with the urine protein content of the control group (1.3±0.18mg / 24h), the urine protein content of the model group (17.1±0.8mg / 24h) was significantly increased (p<0.001); compared with the urine protein content of the model group (17.1±0.8mg / 24h), the urine protein content of the positive drug group (16.0±0.5mg / 24h) was significantly decreased (p<0.01), and the urine protein content of the tetrapeptide FP4 group (9.87±0.46mg / 24h) was also significantly decreased (p<0.001).

[0069] The statistical results of serum creatinine levels in each group of mice are shown in Figure 6 .Depend on Figure 6 It can be seen that compared with the serum creatinine content of the control group (42.17±1.19μM), the serum creatinine content of the model group (135.8±4μM) was significantly increased (p<0.001); compared with the serum creatinine content of the model group (135.8±4μM), the serum creatinine content of the positive drug group (125.3±1.9μM) was significantly decreased (p<0.001), and the serum creatinine content of the tetrapeptide FP4 group (95.66±1.25μM) was also significantly decreased (p<0.001).

[0070] The statistical results of serum urea nitrogen content of mice in each group are shown in Figure 7 .Depend on Figure 7It can be seen that compared with the serum urea nitrogen content of the control group (5.59±0.26mM), the serum urea nitrogen content of the model group (21.2±0.80mM) was significantly increased (p<0.001); compared with the serum urea nitrogen content of the model group (21.2±0.80mM), the serum urea nitrogen content of the positive drug group (19.15±0.56mM) was significantly decreased (p<0.001), and the serum urea nitrogen content of the tetrapeptide FP4 group (14±0.32mM) was also significantly decreased (p<0.001).

[0071] In summary, the active oligopeptide FGVP (tetrapeptide FP4) obtained by screening from Pacific salmon sperm protein hydrolysate in the present invention can significantly reduce the fasting blood glucose, serum creatinine, urea nitrogen and urine protein content of diabetic mice, has the function of alleviating diabetic kidney damage, has better activity and less toxic side effects than the positive drug metformin, and can be used in drugs to alleviate diabetic kidney damage.

[0072] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. Use of a tetrapeptide FP4 having the function of alleviating diabetic kidney damage in the preparation of a drug for alleviating diabetic kidney damage, wherein the amino acid sequence of the tetrapeptide FP4 is FGVP, as shown in SEQ ID NO: 2 in the sequence listing.

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