Sea cucumber-derived dual inhibitory peptide with functions of reducing blood pressure and blood sugar and application of sea cucumber-derived dual inhibitory peptide
Through the dual inhibitory peptide WEPFP prepared from sea cucumber viscera, the side effects and safety issues of existing ACE and DPP-IV inhibitors in the treatment of hypertension and type 2 diabetes are solved, and safer and more effective blood pressure and blood sugar lowering effects are achieved.
Patent Information
- Application Number
- CN202510122808.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-26
- Publication Date
- 2025-04-11
AI Technical Summary
Existing ACE inhibitors and DPP-IV inhibitors have potential side effects and long-term safety issues in the treatment of hypertension and type 2 diabetes, resulting in the search for safer and more effective treatments becoming a hot topic in research.
A double inhibitory peptide from sea cucumber origin, with an amino acid sequence of WEPFP, can inhibit the activity of ACE and DPP-IV at the same time.
This dual inhibitory peptide can effectively lower blood pressure and blood sugar levels, has application value in the preparation of blood pressure and blood sugar-lowering drugs, and has high safety and low side effects due to its natural source.
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Figure CN119798371A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of active peptide biotechnology, and in particular to a sea cucumber-derived dual inhibitory peptide having the functions of lowering blood pressure and blood sugar and an application thereof. Background Art
[0002] Hypertension and type 2 diabetes are major chronic diseases that affect human health worldwide, and their pathogenesis is complex and diverse. Among them, angiotensin-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) are two key enzymes that play a pivotal role in the pathophysiological process of these two diseases. As an important component of the blood pressure regulation system, ACE catalyzes the conversion of angiotensin I to angiotensin II and affects the kallikrein-kinin system (KKS), leading to the degradation of bradykinin, thereby causing blood pressure to rise. This mechanism makes ACE a key target in the treatment of hypertension, and inhibiting its activity has become an effective means to lower blood pressure. On the other hand, DPP-IV plays an important role in regulating the degradation of various bioactive peptides in the body, especially glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), two peptide substances that play a core role in blood sugar regulation. By inhibiting the activity of DPP-IV, the degradation of GLP-1 and GIP can be reduced, their half-life in the body can be prolonged, and the secretion of insulin can be promoted, the release of glucagon can be inhibited, and the purpose of effectively lowering blood sugar can be achieved.
[0003] However, although chemically synthesized ACE inhibitors and DPP-IV inhibitors have achieved significant hypotensive and hypoglycemic effects in clinical practice, their potential side effects and safety issues in long-term use are still worrying. Therefore, finding safer and more effective treatments has become a hot topic in current research. In this regard, ACE and DPP-IV inhibitory functional peptides derived from natural proteins have attracted much attention due to their high safety, low side effects and good biocompatibility. As a high-protein, low-fat marine organism, sea cucumbers are not only rich in a variety of nutrients, such as collagen, sulfated polysaccharides, sea cucumber saponins, and a variety of trace elements, vitamins and minerals, but also their rich protein resources provide great potential for the development of such functional peptides. Summary of the invention
[0004] The purpose of the present invention is to provide a dual inhibitory peptide derived from sea cucumber and having the functions of lowering blood pressure and blood sugar and its application, so as to solve the problems existing in the above-mentioned prior art.
[0005] To achieve the above object, the present invention provides the following solutions:
[0006] One of the technical solutions of the present invention is a dual inhibitory peptide derived from sea cucumber and having the functions of lowering blood pressure and blood sugar, and its amino acid sequence is WEPFP.
[0007] The second technical solution of the present invention is the use of the dual inhibitory peptide in the preparation of angiotensin converting enzyme inhibitors.
[0008] The third technical solution of the present invention is an angiotensin converting enzyme inhibitor, comprising the dual inhibitory peptide.
[0009] A fourth technical solution of the present invention is the use of the dual inhibitory peptide in the preparation of antihypertensive drugs.
[0010] A fifth technical solution of the present invention is a blood pressure lowering drug comprising the dual inhibitory peptide.
[0011] The sixth technical solution of the present invention is the use of the dual inhibitory peptide in the preparation of dipeptidyl peptidase IV inhibitors.
[0012] The seventh technical solution of the present invention is a dipeptidyl peptidase IV inhibitor, comprising the dual inhibitory peptide.
[0013] An eighth technical solution of the present invention is the use of the dual inhibitory peptide in the preparation of hypoglycemic drugs.
[0014] The ninth technical solution of the present invention is a blood sugar lowering drug, comprising the dual inhibitory peptide.
[0015] The technical effect of the present invention compared with the prior art: the present invention prepares and identifies a dual inhibitory peptide derived from sea cucumber viscera with the functions of lowering blood pressure and blood sugar, the dual inhibitory peptide has ACE and DPP-IV dual inhibitory peptides, can simultaneously inhibit the activities of ACE and DPP-IV, and has an alleviating effect on hypertension, type 2 diabetes and related complications, and has application value in the preparation of drugs with the effects of lowering blood pressure and blood sugar. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 The amino acid structure and sequence secondary mass spectrometry identification diagram of the dual inhibitory peptide;
[0017] Figure 2 This is the inhibition rate of dual inhibitory peptides on ACE at different concentrations;
[0018] Figure 3 The schematic diagram of the three-dimensional conformation of the dual inhibitory peptide docking with ACE;
[0019] Figure 4 It is a two-dimensional schematic diagram of the docking force between dual inhibitory peptide and ACE;
[0020] Figure 5 This is a graph showing the inhibition rate of dual inhibitory peptides on DPP-IV at different concentrations;
[0021] Figure 6It is a schematic diagram of the three-dimensional conformation of the dual inhibitory peptide docking with DPP-IV;
[0022] Figure 7 This is a two-dimensional schematic diagram of the docking force between the dual inhibitory peptide and DPP-IV. DETAILED DESCRIPTION
[0023] The technical solution of the present invention is further explained below by way of embodiments, but the protection scope of the present invention is not limited in any form by the embodiments.
[0024] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.
[0025] The embodiment of the present invention provides a dual inhibitory peptide derived from sea cucumber and having the functions of lowering blood pressure and blood sugar, and the amino acid sequence of the peptide is WEPFP.
[0026] The present invention also provides a method for preparing the dual inhibitory peptide, comprising the following steps:
[0027] (1) enzymolyzing the sea cucumber viscera with aminopeptidase and flavor protease to obtain a sea cucumber viscera enzymolysis product;
[0028] (2) After the sea cucumber intestinal viscera hydrolysate is subjected to ultrafiltration and desalting treatment, the peptide sequences in the sea cucumber intestinal viscera hydrolysate are identified by LC-MS / MS liquid phase mass spectrometry to construct a sea cucumber intestinal viscera hydrolysate peptide database, and an enzymatic hydrolysate containing the ACE and DPP-IV dual inhibitory peptide derived from the sea cucumber is obtained;
[0029] (3) Screening out a dual inhibitory peptide of ACE and DPP-IV from sea cucumber.
[0030] In some specific embodiments, the enzymatic hydrolysis conditions are: the usage amount of aminopeptidase is 600U / g, the usage amount of flavor protease is 1000U / g, the usage amount of sea cucumber viscera substrate is 20%, one-time enzymatic hydrolysis is carried out at 50°C for 36h, and the enzymatic hydrolysis pH is 7.
[0031] In some specific embodiments, the screening method is: identifying the molecular weight and sequence of peptides in the sea cucumber visceral hydrolysate, screening the peptide sequence composition in the hydrolysate, and constructing a peptide composition database; further predicting the ACE inhibitory activity and DPP-IV inhibitory activity of the sequences in the peptide database, and screening for potential ACE and DPP-IV dual inhibitory peptides based on the docking energy value and activity score, and analyzing the secondary mass spectrometry structure to obtain the amino acid sequence and structure of the dual inhibitory peptide.
[0032] The embodiment of the present invention also provides the use of the dual inhibitory peptide in the preparation of angiotensin converting enzyme inhibitors.
[0033] The embodiment of the present invention also provides an angiotensin converting enzyme inhibitor, comprising the dual inhibitory peptide.
[0034] The embodiment of the present invention also provides the use of the dual inhibitory peptide in the preparation of blood pressure lowering drugs.
[0035] The embodiment of the present invention also provides a blood pressure lowering drug, comprising the dual inhibitory peptide.
[0036] The embodiment of the present invention also provides the use of the dual inhibitory peptide in the preparation of a dipeptidyl peptidase IV inhibitor.
[0037] The embodiment of the present invention also provides a dipeptidyl peptidase IV inhibitor, comprising the dual inhibitory peptide.
[0038] The embodiment of the present invention also provides the use of the dual inhibitory peptide in the preparation of hypoglycemic drugs.
[0039] The embodiment of the present invention also provides a blood sugar lowering drug, comprising the dual inhibitory peptide.
[0040] The present invention provides a sea cucumber-derived dual inhibitory peptide with the functions of lowering blood pressure and blood sugar. The dual inhibitory peptide can simultaneously inhibit the activities of ACE and DPP-IV, play a role in preventing or even alleviating diseases such as hypertension and hyperglycemia, and has important significance for the actual production and theoretical research of multifunctional inhibitory peptides.
[0041] Example 1: Preparation of dual inhibitory peptides from sea cucumber viscera
[0042] After the sea cucumber viscera were removed, cleaned and homogenized, aminopeptidase and flavor protease were used for composite enzymolysis, wherein the amount of aminopeptidase used was 600U / g, the amount of proline endonuclease used was 1000U / g, the amount of sea cucumber viscera substrate used was 20%, the enzymolysis conditions were one-time enzymolysis at 50°C for 36h, and the pH value of the enzymolysis system was continuously monitored and adjusted to 7 during the enzymolysis process. The final product of the sea cucumber viscera enzymolysis was ultrafiltered and desalted using a solid phase extraction column (SEP-PAK C18 solid phase extraction column, Waters, USA) to prepare a sea cucumber viscera hydrolyzate containing dual inhibitory peptides. LC-MS / MS liquid phase mass spectrometry was further used to determine the molecular weight and sequence of each peptide prepared in the sea cucumber viscera hydrolyzate to form a peptide database of sea cucumber intestinal egg enzymolysis products.
[0043] Example 2: Screening and mining of peptides with dual inhibitory effects on ACE and DPP-IV
[0044] The peptide molecular weight and sequence of the obtained sea cucumber visceral enzymatic hydrolysate were identified by LC-MS / MS liquid phase mass spectrometry to form a peptide database; the peptides were further predicted for ACE and DPP-IV inhibitory activity and function using the PeptideRanker functional activity predictor and the Toixinpred safety and toxicity evaluation predictor, and based on the functional score, potential ACE and DPP-IV dual inhibitory peptides were screened, and their secondary mass spectrometry structures were analyzed to obtain the amino acid sequence and structure of the dual inhibitory peptide. The results are as follows Figure 1 As shown, the secondary mass spectrum of the screened ACE and DPP-IV dual inhibitory peptide shows that the amino acid sequence and arrangement of the peptide is WEPFP.
[0045] Example 3: Verification of ACE inhibitory activity of ACE and DPP-IV dual inhibitory peptide WEPFP
[0046] The ACE inhibitory activity of ACE and DPP-IV dual inhibitory peptide WEPFP was determined using a 96-well plate using a visible spectrophotometer. The ACE inhibition rate was determined using 1mM N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPPG) as a substrate; 0.1U / mL angiotensin converting enzyme (ACE); HEPES as a buffer matrix (1.901g HEPES reagent, 1.755g NaCl reagent, dissolved in an appropriate amount of deionized water, and then adjusted to pH 8.3 with NaOH solution, and fixed to 100mL for standby use). The determination steps are as follows: adding samples of different concentrations, as well as FAPPG substrate, ACE and buffer, etc. in a 96-well plate in sequence; using an ELISA reader to determine the absorbance of the sample system at 340 nm and record it; placing the sample system in a 37°C shaker for incubation for 30 minutes, and again determining the absorbance of the sample system at 340 nm using an ELISA reader after the reaction; the ACE inhibition rate of the sample is determined by calculating the change in absorbance before and after the reaction.
[0047] The calculation formula is: ACE inhibition rate of sample % = 1-change in sample absorbance value / change in blank absorbance value.
[0048] The results are as follows Figure 2 As shown, when the concentration of ACE and DPP-IV dual inhibitory peptide WEPFP was 10 μg / mL, the inhibition rate of ACE was 12.8%; when the concentration of ACE and DPP-IV dual inhibitory peptide WEPFP was 25 μg / mL, the inhibition rate of ACE was 32.0%; when the concentration of ACE and DPP-IV dual inhibitory peptide WEPFP was 50 μg / mL, the inhibition rate of ACE was 34.4%; when the concentration of ACE and DPP-IV dual inhibitory peptide WEPFP was 100 μg / mL, the inhibition rate was 60.86% (see Figure 2 ). According to the results of ACE inhibitory activity assay, when the concentration of ACE and DPP-IV dual inhibitory peptide WEPFP is large enough, it can completely inhibit ACE. The above results show that ACE and DPP-IV dual inhibitory peptide WEPFP has effective ACE inhibitory activity.
[0049] Example 4: Molecular simulation docking of ACE and DPP-IV dual inhibitory peptide WEPFP with ACE
[0050] The ACE and DPP-IV dual inhibitory peptide WEPFP was docked with ACE by molecular simulation to further determine the mechanism of action of the active peptide when it exerts its ACE inhibitory activity. The three-dimensional conformational map of the ACE and DPP-IV dual inhibitory peptide WEPFP was drawn using Discovery studio software, and molecular simulation docking was performed with the catalytic active center of ACE (PDB: 1o8a), and the interaction force between the ACE and DPP-IV dual inhibitory peptide WEPFP and the key amino acid residues in the ACE active center was analyzed based on the docking results.
[0051] The results are as follows Figure 3 As shown in the figure, the dual inhibitory peptide WEPFP for ACE and DPP-IV can bind tightly to the active center of ACE and can be simulated and docked within the normal analysis time. The dual inhibitory peptide WEPFP for ACE and DPP-IV interacts with multiple amino acid residues in the active center of ACE, and the main amino acid residues are His387, Glu384, Glu411, His383, Val379, Val380, Glu376, Phe157, Ala356, Ala354, Gln281, Val518, His353, and Lys511.
[0052] The results of the interaction analysis between the ACE and DPP-IV dual inhibitory peptide WEPFP and the amino acid residues in the active center of ACE are shown in Figure 2. Figure 4 As shown in the figure, the ACE and DPP-IV dual inhibitory peptide WEPFP forms three hydrogen bonds, three salt bridges and mutual attraction, two carbon-hydrogen bonds, one Pi-Anion, two Pi-Pi stacking forces, two alkyl groups and two Pi-alkyl forces with ACE.
[0053] In summary, the dual inhibitory peptide WEPFP for ACE and DPP-IV can bind to ACE mainly through hydrogen bonds, hydrophobic interactions and Zn 2+ The binding effect interacts with the active center residues of ACE and inhibits the activity of ACE. That is, from the perspective of molecular simulation docking, it is further proved that the ACE and DPP-IV dual inhibitory peptide WEPFP has significant ACE inhibitory activity.
[0054] Example 5: Verification of DPP-IV inhibitory activity of ACE and DPP-IV dual inhibitory peptide WEPFP
[0055] The DPP-IV inhibitory activity of the ACE and DPP-IV dual inhibitory peptide WEPFP was determined using a 96-well plate. The DPP-IV inhibition rate was determined using 1.6mM Gly-Pro-pNA as a substrate; 0.1U / mL DPP-IV; Tris-HCl as a buffer matrix (100mM, pH 8.0). The determination steps are as follows: different concentrations of ACE and DPP-IV dual inhibitory peptide WEPFP samples, as well as Gly-Pro-pNA substrate, DPP-IV and buffer were added to the 96-well plate in sequence; the sample system was incubated at 37℃ for 60min and the reaction was terminated. The absorbance value of the sample system at 504nm was determined using an ELISA reader and recorded, and the determined DPP-IV inhibition rate was obtained according to the calculation formula.
[0056] The calculation formula is:
[0057] Among them, A, B, C, and D are the absorbance values of the sample group, control group, sample blank group, and blank group at 504 nm, respectively.
[0058] The results are as follows Figure 4 As shown, when the concentration of ACE and DPP-IV dual inhibitory peptide WEPFP was 100 μg / mL, the inhibition rate of DPP-IV was 21.73%; when the concentration of ACE and DPP-IV dual inhibitory peptide WEPFP was 200 μg / mL, the inhibition rate was 29.28%; when the concentration of ACE and DPP-IV dual inhibitory peptide WEPFP was 400 μg / mL, the DPP-IV inhibition rate was 70.28%; when the concentration of ACE and DPP-IV dual inhibitory peptide WEPFP was 800 μg / mL, the DPP-IV inhibition rate was 97.09% (see Figure 5 ). According to the DPP-IV inhibitory activity assay results, the ACE and DPP-IV dual inhibitory peptide WEPFP has effective DPP-IV inhibitory activity.
[0059] Example 6: Molecular simulation docking of ACE and DPP-IV dual inhibitory peptide WEPFP and DPP-IV
[0060] The ACE and DPP-IV dual inhibitory peptide WEPFP was docked with DPP-IV by molecular simulation to further determine the mechanism of action of the active peptide when it exerts its DPP-IV inhibitory activity. The three-dimensional conformational map of the ACE and DPP-IV dual inhibitory peptide WEPFP was drawn using Discovery studio software, and molecular simulation docking was performed with the catalytic active center of DPP-IV (PDB: 2QT9), and the interaction force between the ACE and DPP-IV dual inhibitory peptide WEPFP and the key amino acid residues in the active center of DPP-IV was analyzed based on the docking results.
[0061] The results are as follows Figure 6 As shown, the ACE and DPP-IV dual inhibitory peptide WEPFP can bind tightly to the DPP-IV active center and can be simulated and docked within the normal analysis time. In addition, the ACE and DPP-IV dual inhibitory peptide WEPFP interacts with multiple amino acid residues in the DPP-IV active center, and the main amino acid residues are Tyr456, Arg471, Val558, and Arg560.
[0062] The results of the interaction force analysis between the dual inhibitory peptide WEPFP for ACE and DPP-IV and the amino acid residues in the active center of DPP-IV are shown in Figure 2. Figure 7 As shown in Figure 3, the ACE and DPP-IV dual inhibitory peptide WEPFP forms 2 hydrogen bond forces, 9 Wandwal forces, 1 salt bridge, and 1 alkyl interaction force with DPP-IV.
[0063] In summary, the ACE and DPP-IV dual inhibitory peptide WEPFP can bind to DPP-IV, and mainly interact with the DPP-IV active center residues through hydrogen bonds, Wandwala forces, salt bridges and alkyl forces to inhibit the activity of DPP-IV. That is, from the perspective of molecular simulation docking, it is further proved that the ACE and DPP-IV dual inhibitory peptide WEPFP has DPP-IV inhibitory activity.
[0064] In summary, the present invention prepares and identifies a dual inhibitory peptide of ACE and DPP-IV, WEPFP, from sea cucumber viscera by a composite enzyme hydrolysis method. The present invention obtains its amino acid sequence and structure by performing secondary mass spectrometry analysis on the active peptide, verifies the inhibitory activity of the dual inhibitory peptide of ACE and DPP-IV on ACE and DPP-IV, and docks the dual inhibitory peptide of ACE and DPP-IV with the active centers of ACE and DPP-IV respectively by molecular simulation docking to illustrate its inhibitory mechanism on the activity of ACE and DPP-IV.
[0065] The invention provides a sea cucumber-derived ACE and DPP-IV dual inhibitory peptide WEPFP, which has ACE and DPP-IV inhibitory activity and has certain blood pressure lowering and blood sugar lowering effects.
[0066] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.
Claims
1. A dual inhibitory peptide derived from sea cucumber with the function of lowering blood pressure and blood sugar, characterized in that: The amino acid sequence of the dual inhibitory peptide is WEPFP.
2. An angiotensin converting enzyme inhibitor, characterized in that The angiotensin converting enzyme inhibitor comprises the dual inhibitory peptide according to claim 1.
3. A blood pressure lowering drug, characterized in that: The blood pressure lowering drug comprises the dual inhibitory peptide according to claim 1.
4. A dipeptidyl peptidase IV inhibitor, characterized in that The dipeptidyl peptidase IV inhibitor comprises the dual inhibitory peptide according to claim 1.
5. A blood sugar lowering drug, characterized in that: The blood sugar lowering drug comprises the dual inhibitory peptide according to claim 1.
6. A use of the dual inhibitory peptide according to claim 1, characterized in that: The dual inhibitory peptide according to claim 1 is used to prepare angiotensin converting enzyme inhibitors, antihypertensive drugs, dipeptidyl peptidase IV inhibitors and hypoglycemic drugs.