Novel ACE inhibitory peptide with DPP-IV inhibitory activity and application thereof

Abstract

The invention discloses a novel ACE (Angiotensin Converting Enzyme) inhibitory peptide with DPP-IV (Dipeptidyl Peptidase-IV) inhibitory activity and application thereof, and belongs to the technical field of bioactive peptide biology. The amino acid sequence of the gene is as shown in SEQ ID NO. 1. The novel ACE inhibitory peptide with DPP-IV inhibitory activity is prepared and identified from sea cucumber intestine egg enzymatic hydrolysate, the ACE activity can be remarkably inhibited through a competitive inhibition mode, the gastrointestinal tract digestion environment is tolerated, meanwhile, the DPP-IV inhibitory activity is achieved, the effect of relieving hypertension and type 2 diabetes mellitus is achieved at the same time, and the ACE inhibitory peptide has the advantages of being safe and reliable. Compared with the prior art, the traditional Chinese medicine composition has the advantages of being simple in structure, safe, high in activity and the like, can play a role in nutrition and health care, is expected to provide effective active ingredients for developing food and products which are free of side effects and have the effects of reducing blood pressure and blood sugar or have the effects of reducing blood pressure and blood sugar, and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the field of active peptide biotechnology, and in particular to a novel ACE inhibitory peptide with DPP-IV inhibitory activity and application thereof. Background Art

[0002] Hypertension and type 2 diabetes mellitus (T2DM) are two common chronic diseases that often coexist, posing a significant threat and burden to global health issues. The metabolic disorder of diabetes is characterized by elevated blood glucose levels, and hypertension is often one of its complications and comorbidities. Both diseases share some underlying pathophysiological mechanisms, such as inflammation, oxidative stress, and endothelial dysfunction, which drive their progression and exacerbate the associated cardiovascular risks. The incidence of cardiovascular disease in patients with diabetes is 2-3 times that of the general population. Traditional treatments usually rely on a combination of drugs to control blood pressure and blood glucose levels, which have significant side effects and often lack multifunctional efficacy. Inhibitors with dual effects of lowering blood pressure and blood glucose can effectively alleviate the impact of complications. Therefore, the discovery of natural bioactive compounds that can simultaneously treat hypertension and hyperglycemia has received increasing attention.

[0003] It is well known that ACE inhibitors have a blood pressure-lowering effect because they interfere with the renin-angiotensin system, promote vasodilation, and reduce vascular resistance. DPP-IV is responsible for the cleavage and inactivation of the incretin hormones glucagon-like peptide-1 and glucose-dependent insulin polypeptide. At the same time, DPP-IV inhibitors play a key role in blood glucose management by increasing incretin levels, thereby enhancing insulin secretion and improving glucose tolerance. Exploring ACE inhibitory peptides that simultaneously exhibit DPP-IV inhibitory activity may provide a new approach for the treatment of hypertension and hyperglycemia, especially for patients with metabolic disorders. Therefore, bioactive peptides with angiotensin-converting enzyme (ACE) inhibition and dipeptidyl peptidase-IV (DPP-IV) inhibition activities are currently a hot topic of research.

[0004] As an important marine biological resource, sea cucumbers have long been widely recognized and used because of their rich variety of effective ingredients and potential value in the field of disease prevention and treatment. However, during the processing of sea cucumbers, their internal organs are often discarded as waste. Recent studies have shown that sea cucumber waste materials may be an important source of functional substances, which can not only facilitate the development of active ingredients, but also greatly increase the high-value utilization of sea cucumber by-products.

[0005] Therefore, how to further extract available active ingredients from the discarded raw materials of sea cucumbers is an urgent problem that needs to be solved. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a novel ACE inhibitory peptide having DPP-IV inhibitory activity and application thereof, so as to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the technical solution of the present invention is as follows: One of the technical solutions of the present invention is a novel ACE inhibitory peptide with DPP-IV inhibitory activity, the amino acid sequence of the inhibitory peptide is shown in SEQ ID NO.1.

[0008] The second technical solution of the present invention is a method for preparing the ACE inhibitory peptide, comprising the following steps: (1) enzymatically hydrolyzing the sea cucumber intestines and eggs using papain and trypsin to obtain a sea cucumber intestines and eggs enzymatic hydrolysis product; (2) After the obtained sea cucumber intestinal egg hydrolysate is subjected to ultrafiltration and desalting treatment, the sequence of the peptide fragments in the sea cucumber intestinal egg hydrolysate is identified by LC-MS / MS liquid chromatography-mass spectrometry to obtain an enzymatic hydrolysate containing the novel ACE inhibitory peptide with DPP-IV inhibitory activity; (3) Screening out new ACE inhibitory peptides with DPP-IV inhibitory activity.

[0009] Furthermore, in step (1), the enzymatic hydrolysis conditions are: the amount of papain used is 1000 U / g (based on the mass of the sea cucumber intestinal egg substrate), the amount of trypsin used is 800 U / g (based on the mass of the sea cucumber intestinal egg substrate), the amount of sea cucumber intestinal eggs used is 15% (the mass fraction of the substrate, i.e., the sea cucumber intestinal eggs in the entire reaction system, and the system contains, in addition to the sea cucumber intestinal eggs, the amount of solvent such as water), the enzymatic hydrolysis is carried out at 65°C for 48 h, and the enzymatic hydrolysis pH is 7.5.

[0010] Furthermore, in step (3), the screening is specifically: Screening out peptide sequences with a molecular weight less than 2 kDa from the enzymatic hydrolysate containing the novel ACE inhibitory peptide having DPP-IV inhibitory activity; The ACE inhibitory activity and DPP-IV inhibitory activity of the screened peptide sequences were predicted, and based on the docking energy value and activity score with ACE, ACE inhibitory peptides with potential DPP-IV inhibitory activity were screened, and their secondary mass spectrometry structures were analyzed to obtain the amino acid sequence and structure of the ACE inhibitory peptide.

[0011] The third technical solution of the present invention is the use of the ACE inhibitory peptide in any of the following: 1) Application in the preparation of angiotensin converting enzyme inhibitors; 2) Application in the preparation of antihypertensive drugs; 3) Application in the preparation of dipeptidyl peptidase IV inhibitors; 4) Application in the preparation of hypoglycemic drugs.

[0012] A fourth technical solution of the present invention is a DPP-IV inhibitory drug, comprising the novel ACE inhibitory peptide described above.

[0013] A fifth technical solution of the present invention is an ACE inhibitory drug, comprising the novel ACE inhibitory peptide described above.

[0014] The sixth technical solution of the present invention is a drug for simultaneously inhibiting DPP-IV and ACE, comprising the novel ACE inhibitory peptide described above.

[0015] The seventh technical solution of the present invention is a blood pressure lowering drug, comprising the novel ACE inhibitory peptide described above.

[0016] The eighth technical solution of the present invention is a blood sugar lowering drug, comprising the novel ACE inhibitory peptide described above.

[0017] The beneficial effects of the present invention include at least: The present invention prepares and identifies a novel ACE inhibitory peptide with DPP-IV inhibitory activity from sea cucumber intestines and eggs. The peptide can significantly inhibit the activity of ACE through a competitive inhibition mode, has gastrointestinal digestion tolerance, and also has DPP-IV inhibitory activity, which has a relieving effect on hypertension, type 2 diabetes and related complications, and has application value in the preparation of products with blood pressure and blood sugar lowering effects.

[0018] The ACE inhibitory peptide involved in the present invention can significantly inhibit the activity of ACE through a competitive inhibition mode, has gastrointestinal digestion tolerance, and also has DPP-IV inhibitory activity. It has the characteristics of simple structure, safety, strong activity, etc., can play a role in nutrition and health care, and is expected to provide effective active ingredients for the development of food and blood pressure lowering and blood pressure lowering efficacy products without side effects, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the amino acid structure and sequence secondary mass spectrometry identification diagram of a new ACE inhibitory peptide with DPP-IV inhibitory activity.

[0020] Figure 2 Schematic diagram of the three-dimensional conformation of the docking of GFPGLP, a new ACE inhibitory peptide with DPP-IV inhibitory activity, with ACE.

[0021] Figure 3 This is a two-dimensional schematic diagram of the docking force between ACE and GFPGLP, a new ACE inhibitory peptide with DPP-IV inhibitory activity.

[0022] Figure 4This is a graph showing the inhibition rate of ACE at different concentrations of GFPGLP, a new ACE inhibitory peptide with DPP-IV inhibitory activity.

[0023] Figure 5 This is the inhibition pattern diagram of a new ACE inhibitory peptide GFPGLP with DPP-IV inhibitory activity.

[0024] Figure 6 This is a graph showing the results of a stability study of a new ACE inhibitory peptide GFPGLP with DPP-IV inhibitory activity in gastric digestive juice.

[0025] Figure 7 The stability test results of a new ACE inhibitory peptide GFPGLP with DPP-IV inhibitory activity in intestinal digestive juice Figure 8 This is a graph showing the inhibition rate of DPP-IV at different concentrations of GFPGLP, a new ACE inhibitory peptide with DPP-IV inhibitory activity.

[0026] Fig. 9 Schematic diagram of the three-dimensional conformation of the docking of a new ACE inhibitory peptide GFPGLP with DPP-IV inhibitory activity and DPP-IV.

[0027] Fig.10 This is a two-dimensional schematic diagram of the docking force between a new ACE inhibitory peptide GFPGLP with DPP-IV inhibitory activity and DPP-IV. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in the field can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0030] The embodiment of the present invention provides a novel ACE inhibitory peptide having DPP-IV inhibitory activity, and its amino acid sequence is shown in SEQ ID NO.1.

[0031] The present invention also provides a method for preparing the ACE inhibitory peptide, comprising the following steps: (1) enzymatically hydrolyzing the sea cucumber intestines and eggs using papain and trypsin to obtain a sea cucumber intestines and eggs enzymatic hydrolysis product; (2) After the sea cucumber intestinal egg enzymatic hydrolysate is subjected to ultrafiltration and desalting treatment, the sequence of the peptide segment in the sea cucumber intestinal egg enzymatic hydrolysate is identified by LC-MS / MS liquid phase mass spectrometry to obtain an enzymatic hydrolysate containing the novel ACE inhibitory peptide with DPP-IV inhibitory activity; (3) Screening out new ACE inhibitory peptides with DPP-IV inhibitory activity.

[0032] In some specific embodiments, the enzymatic hydrolysis conditions are: the dosage of papain is 1000U / g, the dosage of trypsin is 800U / g, the dosage of sea cucumber viscera substrate is 15%, the enzymatic hydrolysis is carried out at 65°C for 48h, and the enzymatic hydrolysis pH is 7.5.

[0033] In some specific embodiments, the screening method is: identifying the molecular weight and sequence of peptides in the sea cucumber intestinal egg enzymatic hydrolysate, and screening peptide sequences with a molecular weight of XXX; further predicting the ACE inhibitory activity and DPP-IV inhibitory activity of the screened peptide sequences, and based on the docking energy value and activity score with ACE, screening for ACE inhibitory peptides with potential DPP-IV inhibitory activity, and analyzing their secondary mass spectrometry structure to obtain the amino acid sequence and structure of the ACE inhibitory peptide.

[0034] The embodiment of the present invention also provides the use of the ACE inhibitory peptide in the preparation of angiotensin converting enzyme inhibitors.

[0035] The embodiment of the present invention further provides an angiotensin converting enzyme inhibitor, comprising the ACE inhibitory peptide.

[0036] The embodiment of the present invention also provides the use of the ACE inhibitory peptide in the preparation of a blood pressure lowering drug.

[0037] The embodiment of the present invention also provides a blood pressure lowering drug, comprising the ACE inhibitory peptide.

[0038] The embodiment of the present invention also provides the use of the ACE inhibitory peptide in the preparation of a dipeptidyl peptidase IV inhibitor.

[0039] The embodiment of the present invention also provides a dipeptidyl peptidase IV inhibitor, comprising the ACE inhibitory peptide.

[0040] The embodiment of the present invention also provides the use of the ACE inhibitory peptide in the preparation of a hypoglycemic drug.

[0041] The embodiment of the present invention also provides a blood sugar lowering drug, comprising the ACE inhibitory peptide.

[0042] The novel ACE inhibitory peptide with DPP-IV inhibitory activity provided by the present invention can significantly inhibit the activity of ACE through competitive inhibition, has gastrointestinal digestion tolerance, can also effectively inhibit the activity of DPP-IV, and plays a role in preventing and even alleviating diseases such as hypertension and hyperglycemia, which is of great significance for the actual production and theoretical research of multifunctional active peptides.

[0043] The following specifically illustrates the solution proposed by the present invention through specific embodiments:

[0044] Example 1 Preparation of ACE inhibitory peptide from sea cucumber intestine and egg After pretreatment such as removal, washing and homogenization of sea cucumber intestine and egg, it was subjected to composite enzymatic hydrolysis using papain and trypsin. The dosage of papain was 1000 U / g, the dosage of trypsin was 800 U / g, the substrate dosage of sea cucumber intestine and egg was 15%, and enzymatic hydrolysis was carried out at 65 °C for 48 h. During the enzymatic hydrolysis process, the pH value of the enzymatic hydrolysis system was continuously monitored and adjusted to 7.5. The final product of enzymatic hydrolysis of sea cucumber intestine and egg was subjected to ultrafiltration treatment and desalted using a solid-phase extraction column (SEP-PAK C18 solid-phase extraction column, Waters, USA) to prepare a sea cucumber viscera hydrolysate containing ACE inhibitory peptide. Further, LC-MS / MS liquid chromatography-mass spectrometry was used to determine the molecular weights and sequences of the peptide segments obtained from the enzymatic hydrolysis of sea cucumber intestine and egg.

[0045] Example 2 Screening and discovery of ACE inhibitory peptide GFPGLP According to the molecular weights and sequences of the peptide segments in the enzymatic hydrolysis product of sea cucumber intestine and egg identified by LC-MS / MS liquid chromatography-mass spectrometry, peptide segment sequences with molecular weights less than 2 kDa were screened to form a peptide segment database, with a total of 1039; the peptide ranker tool (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used to predict the functional scores of the peptide segments, and the predicted activity scores were sorted; further, the ToxinPred tool (https: / / crdd.osdd.net / raghava / toxinpred / ) was used to analyze the toxicity of the above peptide segments to ensure that the peptide segments screened subsequently were non-toxic. Further, 50 peptide segments with the top 50 predicted non-toxic and activity scores (all predicted non-toxic) were subjected to molecular simulation docking, and ACE inhibitory peptides with potential functional activities were screened based on the docking energy values.

[0046] The screened ACE inhibitory peptides with potential functional activity were further docked with ACE by molecular simulation, and the three-dimensional conformational map of the ACE inhibitory peptide was drawn using Discovery studio software, and molecular simulation docking was performed with the catalytic active center of ACE (PDB: 1o8a). Based on the docking results, the energy value required for the docking of the ACE inhibitory peptide with ACE was analyzed, and the ACE inhibitory peptides with small required energy values ​​and priority functional scores were selected, and their secondary mass spectrometry structures were analyzed to obtain the amino acid sequence and structure of the ACE inhibitory peptide. The results are as follows Figure 1 As shown, the secondary mass spectrum of the screened active peptide shows that the amino acid sequence of the active peptide is SEQ ID NO.1: GFPGLP.

[0047] Example 3 Analysis of the binding interaction between ACE inhibitory peptide GFPGLP and ACE Based on the molecular simulation docking results in the screening process, the binding site and force type between the screened ACE inhibitory peptide GFPGLP and ACE were further analyzed to analyze its potential mechanism of action when exerting its ACE inhibitory activity.

[0048] The results are as follows Figure 2 As shown, the ACE inhibitory peptide GFPGLP can bind tightly to the ACE active center and can be simulated and docked within the normal analysis time. The ACE inhibitory peptide GFPGLP interacts with multiple amino acid residues in the ACE active center, and the main amino acid residues are Lys368, Asp377, Tyr523, Val518, Glu162, His383, His387, Glu384, Gln281, Ala354, His513, and His353.

[0049] The results of the interaction analysis between the ACE inhibitory peptide GFPGLP and the amino acid residues in the active center of ACE are shown in Figure 2. Figure 3 As shown. The ACE inhibitory peptide GFPGLP forms three hydrogen bond forces with ACE, three interactions between alkyl and π-alkyl, one π-π interaction, two carbon-hydrogen forces, three salt bridges or mutual attraction, and Zn 2+ Combination effect.

[0050] In summary, the ACE inhibitory peptide GFPGLP 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 thus inhibits the activity of ACE.

[0051] Example 4 Verification of ACE inhibitory activity of ACE inhibitory peptide GFPGLP The inhibitory activity of ACE inhibitory peptide GFPGLP on ACE was determined using a 96-well plate using a visible spectrophotometer. The ACE inhibition rate was determined using 1 mM N-[3-(2-furyl) acryloyl]-L-phenylalanylglycylglycine (FAPPG) as a substrate, 0.1 U / mL angiotensin converting enzyme (ACE), and HEPES as a buffer matrix (1.901 g HEPES reagent and 1.755 g NaCl reagent were weighed and dissolved in an appropriate amount of deionized water, and then the buffer pH was adjusted to 8.3 with NaOH solution, and the volume was fixed to 100 mL for standby use). The determination steps are as follows: adding different concentrations of ACE inhibitory peptide GFPGLP samples, as well as FAPPG substrate, ACE and buffer, etc. to a 96-well plate in sequence; using an enzyme 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 shaking and incubation for 30 minutes, and the absorbance of the sample system after the reaction at 340 nm is determined again using an enzyme reader; the ACE inhibition rate of the sample is determined by calculating the change in absorbance before and after the reaction.

[0052] The calculation formula is: ACE inhibition rate of sample % = (1-change in sample absorbance) / change in blank absorbance * 100%.

[0053] The results are as follows Figure 4 As shown, when the concentration of the ACE inhibitory peptide GFPGLP is 10 μg / mL, the inhibition rate of the ACE inhibitory peptide GFPGLP on ACE is 27.0%; when the concentration of the ACE inhibitory peptide GFPGLP is 25 μg / mL, the inhibition rate of ACE is 58.62%; when the concentration of the ACE inhibitory peptide GFPGLP is 50 μg / mL, the inhibition rate of ACE is 82.74%; when the concentration of the ACE inhibitory peptide GFPGLP is 100 μg / mL, the inhibition rate of the ACE inhibitory peptide GFPGLP is 96.28%; when the concentration of the ACE inhibitory peptide GFPGLP continues to increase, the inhibition rate of ACE can reach 100% (see Figure 4 ). According to the results of ACE inhibitory activity assay, when the concentration of ACE inhibitory peptide GFPGLP is greater than 100 μg / mL, it can completely inhibit ACE. The above results show that ACE inhibitory peptide GFPGLP has significant ACE inhibitory activity.

[0054] Example 5 Study on the ACE inhibition mode of ACE inhibitory peptide GFPGLP The ACE inhibition mode of the ACE inhibitory peptide GFPGLP was analyzed by measuring the ACE enzyme inhibition kinetics of different concentrations of ACE inhibitory peptide GFPGLP samples under different concentrations of substrates. The ACE inhibitory peptide GFPGLP samples were diluted to 200 and 400 μg / mL for standby use, and the substrate FAPPG was diluted to 0.5, 1, and 1.5 mM concentrations for standby use. Different concentrations of ACE inhibitory peptide GFPGLP were used to prepare each reaction system with different concentrations of FAPPG substrates, and their ACE inhibition rates were determined. The inhibition kinetics of ACE were analyzed according to the enzyme double reciprocal plotting method and the Michel equation, and then the ACE inhibition mode of the ACE inhibitory peptide GFPGLP was analyzed.

[0055] The results are as follows Figure 5 As shown in Figure 2, with the increase of the concentration of ACE inhibitory peptide GFPGLP, the maximum reaction rate V max No change, K m The value increased, indicating that the ACE inhibitory peptide GFPGLP was in a competitive inhibition mode.

[0056] Example 6 Gastrointestinal digestive enzyme stability of ACE inhibitory peptide GFPGLP The ACE inhibitory peptide GFPGLP sample was diluted to its IC for ACE inhibition. 50 The concentration of 1M HCl solution was used to adjust the pH of the system to 2, and 2% pepsin was added to prepare simulated gastric digestive fluid. The ACE inhibitory peptide GFPGLP sample was mixed with the pepsin solution and incubated at 37°C. After the reaction, samples were taken every 30 minutes to determine the ACE inhibition rate, and the treatment was carried out for 3 hours to investigate the stability of the ACE inhibitory peptide GFPGLP in gastric digestive fluid. Subsequently, 1M NaOH was used to adjust the pH to 7.5 to inactivate the enzymatic hydrolysis product, and the gastric hydrolyzate was obtained for trypsin treatment for 3 hours. After the start of trypsin treatment, samples were taken every 30 minutes and the samples were inactivated at 95°C for 10 minutes, cooled to 37°C, and the in vitro ACE inhibition rate was determined to analyze its stability in intestinal digestive fluid.

[0057] The results are as follows Figure 6 As shown, the concentration of ACE inhibitory peptide GFPGLP is its IC for ACE inhibition 50 At 1.544 mg / mL, after being treated with the above-mentioned gastric digestive juice, the inhibition rate of the digested ACE inhibitory peptide GFPGLP on ACE changed from 49.26% to 40.36%, with a small decrease in inhibition rate, and still having good ACE inhibitory activity, indicating that the ACE inhibitory peptide GFPGLP has good gastric digestive juice tolerance. After being treated with intestinal digestive juice for 3 hours, its inhibition rate on ACE finally changed to 40.07%, indicating that the ACE inhibitory peptide GFPGLP has good intestinal digestive juice tolerance (see Figure 7 ).

[0058] Example 7 Verification of DPP-IV Inhibitory Activity of ACE Inhibitory Peptide GFPGLP The DPP-IV inhibitory activity of ACE inhibitory peptide GFPGLP was determined using a 96-well plate. The DPP-IV inhibition rate was determined using 1.6mM Gly-Pro-pNA as substrate; 0.1U / mL angiotensin converting enzyme (ACE); and Tris-HCl as a buffer matrix (100mM, pH8.0). The determination steps were as follows: different concentrations of ACE inhibitory peptide GFPGLP 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.

[0059] The calculation formula is: ; 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.

[0060] The results are as follows Figure 8 As shown, when the concentration of the ACE inhibitory peptide GFPGLP is 100 μg / mL, the inhibition rate of the ACE inhibitory peptide GFPGLP on DPP-IV is 48.08%; when the concentration of the ACE inhibitory peptide GFPGLP is 200 μg / mL, the inhibition rate of the DPP-IV is 89.56%; when the concentration of the ACE inhibitory peptide GFPGLP is 400 μg / mL, the inhibition rate of the DPP-IV is 92.46%; when the concentration of the ACE inhibitory peptide GFPGLP is 800 μg / mL, the inhibition rate of the ACE inhibitory peptide GFPGLP on DPP-IV is 96.73% (see Figure 8 ). According to the results of DPP-IV inhibitory activity assay, the ACE inhibitory peptide GFPGLP has significant DPP-IV inhibitory activity.

[0061] Example 8 Molecular simulation docking of ACE inhibitory peptide GFPGLP and DPP-IV The ACE inhibitory peptide GFPGLP 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 inhibitory peptide GFPGLP 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 inhibitory peptide GFPGLP and the key amino acid residues in the active center of DPP-IV was analyzed based on the docking results.

[0062] The results are as follows Fig. 9 As shown, the ACE inhibitory peptide GFPGLP can bind tightly to the active center of DPP-IV and can be simulated and docked within the normal analysis time. The active ACE inhibitory peptide GFPGLP interacts with multiple amino acid residues in the active center of DPP-IV, and the main amino acid residues are Arg471, Arg125, His126, Ser209, Glu205, Glu206, Tyr666, Phe357, Arg358, Val207, Arg669, and Leu57.

[0063] The results of the interaction force analysis between the ACE inhibitory peptide GFPGLP and the amino acid residues in the active center of DPP-IV are shown in Figure 2. Fig.10 As shown in Figure 3, the ACE inhibitory peptide GFPGLP forms three hydrogen bonding forces, seven van der Waals forces, three salt bridges and mutual attraction, and one Pi-Pi stacking force with DPP-IV.

[0064] In summary, the ACE inhibitory peptide GFPGLP can bind to DPP-IV, and mainly interacts with the DPP-IV active center residues through hydrogen bonds, van der Waals forces and salt bridges to inhibit the activity of DPP-IV. That is, from the perspective of molecular simulation docking, it is further proved that the ACE inhibitory peptide GFPGLP has significant DPP-IV inhibitory activity.

[0065] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0066] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0067] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A novel ACE inhibitory peptide having DPP-IV inhibitory activity, characterized in that: The amino acid sequence of the inhibitory peptide is shown in SEQ ID NO.

1.

2. The method for preparing the ACE inhibitory peptide according to claim 1, characterized in that: The method comprises the following steps: (1) enzymatically hydrolyzing the sea cucumber intestines and eggs using papain and trypsin to obtain a sea cucumber intestines and eggs enzymatic hydrolysis product; (2) After the obtained sea cucumber intestinal egg hydrolysate is subjected to ultrafiltration and desalting treatment, the sequence of the peptide fragments in the sea cucumber intestinal egg hydrolysate is identified by LC-MS / MS liquid chromatography-mass spectrometry to obtain an enzymatic hydrolysate containing the novel ACE inhibitory peptide with DPP-IV inhibitory activity; (3) Screening out new ACE inhibitory peptides with DPP-IV inhibitory activity.

3. The method according to claim 2, characterized in that In step (1), the enzymatic hydrolysis conditions are: the amount of papain used is 1000 U / g, the amount of trypsin used is 800 U / g, the amount of sea cucumber intestines and eggs used is 15%, the enzymatic hydrolysis is carried out at 65°C for 48 hours, and the enzymatic hydrolysis pH is 7.

5.

4. The method according to claim 2, characterized in that: In step (3), the screening is specifically as follows: Screening out peptide sequences with a molecular weight less than 2 kDa from an enzymatic hydrolyzate containing the novel ACE inhibitory peptide having DPP-IV inhibitory activity; The ACE inhibitory activity and DPP-IV inhibitory activity of the screened peptide sequences were predicted, and based on the docking energy value and activity score with ACE, ACE inhibitory peptides with potential DPP-IV inhibitory activity were screened, and their secondary mass spectrometry structures were analyzed to obtain the amino acid sequence and structure of the ACE inhibitory peptide.

5. Use of the ACE inhibitory peptide according to claim 1 in any of the following: 1) Application in the preparation of angiotensin converting enzyme inhibitors; 2) Application in the preparation of antihypertensive drugs; 3) Application in the preparation of dipeptidyl peptidase IV inhibitors; 4) Application in the preparation of hypoglycemic drugs.

6. A DPP-IV inhibitory drug, characterized in that: The invention comprises the novel ACE inhibitory peptide according to claim 1.

7. An ACE inhibitory drug, characterized in that: The invention comprises the novel ACE inhibitory peptide according to claim 1.

8. A drug for inhibiting both DPP-IV and ACE, characterized in that: The invention comprises the novel ACE inhibitory peptide according to claim 1.

9. A blood pressure lowering drug, characterized in that: The invention comprises the novel ACE inhibitory peptide according to claim 1.

10. A hypoglycemic drug, characterized in that: The invention comprises the novel ACE inhibitory peptide according to claim 1.

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