An ACE inhibitory peptide with DPP-IV inhibitory activity and its application

Abstract

The present invention discloses an ACE inhibitory peptide with DPP-IV inhibitory activity and its application, belonging to the field of active peptide biotechnology; its amino acid sequence is shown in SEQ ID NO.1. The present invention prepares and identifies an ACE inhibitory peptide with DPP-IV inhibitory activity from sea cucumber intestinal egg hydrolysate, which can significantly inhibit the activity of ACE through a competitive inhibition mode, tolerate the gastrointestinal digestive environment, and also has DPP-IV inhibitory activity, and has a mitigating effect on hypertension and type 2 diabetes. 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 side-effect-free blood pressure-lowering, blood sugar-lowering or blood pressure-lowering and blood sugar-lowering efficacy products, etc., and has broad application prospects.

Description

Technical Field

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

[0002] Hypertension and type 2 diabetes mellitus (T2DM) are two common chronic diseases that frequently coexist, posing a significant threat and burden to global health. Diabetes mellitus is characterized by metabolic derangements characterized by elevated blood glucose levels, and hypertension is often a comorbidity and comorbidity. Both diseases share underlying pathophysiological mechanisms, such as inflammation, oxidative stress, and endothelial dysfunction, that drive their progression and exacerbate the associated cardiovascular risks. The incidence of cardiovascular disease in patients with diabetes is two to three times higher than in the general population. Traditional treatments typically rely on drug combinations to control both blood pressure and blood glucose levels, but these therapies have significant side effects and often lack multifunctional efficacy. Inhibitors with dual blood pressure and blood glucose lowering effects could effectively mitigate the impact of these complications. Therefore, the discovery of natural bioactive compounds that can simultaneously treat hypertension and hyperglycemia has attracted increasing attention.

[0003] ACE inhibitors are well known to lower blood pressure by interfering with the renin-angiotensin system, promoting vasodilation and reducing vascular resistance. DPP-IV is responsible for the cleavage and inactivation of the incretin hormones glucagon-like peptide-1 and glucose-dependent insulin polypeptide. Simultaneously, DPP-IV inhibitors play a key role in blood glucose management by increasing incretin levels, thereby enhancing insulin secretion and improving glucose tolerance. The discovery of ACE-inhibitory peptides that also exhibit DPP-IV inhibitory activity may provide a new approach for the treatment of hypertension and hyperglycemia, particularly in patients with metabolic disorders. Therefore, bioactive peptides that inhibit both angiotensin-converting enzyme (ACE) and dipeptidyl peptidase-IV (DPP-IV) are currently a hot topic of research.

[0004] As an important marine biological resource, sea cucumbers have long been widely recognized and utilized for their rich variety of therapeutic ingredients and potential value in disease prevention and treatment. However, during the processing of sea cucumbers, their internal organs are often discarded as waste. Recent research indicates that sea cucumber waste materials may be an important source of functional compounds, not only facilitating the development of active ingredients but also significantly increasing 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 an ACE inhibitory peptide with DPP-IV inhibitory activity and its application, 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:

[0008] One of the technical solutions of the present invention is an ACE inhibitory peptide with DPP-IV inhibitory activity, the amino acid sequence of the inhibitory peptide is shown in SEQ ID NO.1.

[0009] The second technical solution of the present invention is a method for preparing the ACE inhibitory peptide, comprising the following steps:

[0010] (1) Enzymatically hydrolyzing the sea cucumber intestines and eggs using papain and trypsin to obtain a sea cucumber intestines and eggs enzymatic hydrolysis product;

[0011] (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 ACE inhibitory peptide with DPP-IV inhibitory activity;

[0012] (3) Screen out ACE inhibitory peptides with DPP-IV inhibitory activity.

[0013] Furthermore, in step (1), the enzymatic hydrolysis conditions are as follows: 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 egg used is 15% (the mass fraction of the substrate, i.e., the sea cucumber intestinal egg in the entire reaction system, and the system contains, in addition to the sea cucumber intestinal egg, 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.

[0014] Furthermore, in step (3), the screening is specifically as follows:

[0015] screening out peptide sequences with a molecular weight of less than 2 kDa from an enzymatic hydrolyzate containing the ACE inhibitory peptide having DPP-IV inhibitory activity;

[0016] 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. Their secondary mass spectrometry structures were analyzed to obtain the amino acid sequence and structure of the ACE inhibitory peptides.

[0017] The third technical solution of the present invention is the use of the ACE inhibitory peptide in any of the following:

[0018] 1) Application in the preparation of angiotensin-converting enzyme inhibitors;

[0019] 2) Application in the preparation of antihypertensive drugs;

[0020] 3) Application in the preparation of dipeptidyl peptidase IV inhibitors;

[0021] 4) Application in the preparation of hypoglycemic drugs.

[0022] A fourth technical solution of the present invention is a DPP-IV inhibitory drug comprising the above-mentioned ACE inhibitory peptide.

[0023] A fifth technical solution of the present invention is an ACE inhibitory drug comprising the above-mentioned ACE inhibitory peptide.

[0024] The sixth technical solution of the present invention is a drug for simultaneously inhibiting DPP-IV and ACE, comprising the ACE inhibitory peptide described above.

[0025] The seventh technical solution of the present invention is a blood pressure lowering drug comprising the above-mentioned ACE inhibitory peptide.

[0026] The eighth technical solution of the present invention is a hypoglycemic drug comprising the ACE inhibitory peptide described above.

[0027] The beneficial effects of the present invention include at least:

[0028] The present invention prepares and identifies an 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. It has an alleviating 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.

[0029] 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, and strong activity. It 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

[0030] Figure 1 This is the amino acid structure and sequence secondary mass spectrometry identification diagram of an ACE inhibitory peptide with DPP-IV inhibitory activity.

[0031] Figure 2 Schematic diagram of the three-dimensional conformation of the docking of ACE inhibitory peptide GFPGLP with DPP-IV inhibitory activity and ACE.

[0032] Figure 3 This is a two-dimensional schematic diagram of the docking interaction between ACE-inhibitory peptide GFPGLP with DPP-IV inhibitory activity and ACE.

[0033] Figure 4 This is a graph showing the inhibition rate of ACE at different concentrations of GFPGLP, an ACE inhibitory peptide with DPP-IV inhibitory activity.

[0034] Figure 5 This is an inhibition pattern diagram of GFPGLP, an ACE inhibitory peptide with DPP-IV inhibitory activity.

[0035] Figure 6 This figure shows the results of the stability study of GFPGLP, an ACE inhibitory peptide with DPP-IV inhibitory activity, in gastric digestive juice.

[0036] Figure 7 Figure 2 shows the stability test results of GFPGLP, an ACE inhibitory peptide with DPP-IV inhibitory activity, in intestinal digestive fluid.

[0037] Figure 8 This is a graph showing the inhibition rate of DPP-IV at different concentrations of GFPGLP, an ACE inhibitory peptide with DPP-IV inhibitory activity.

[0038] Figure 9 Schematic diagram of the three-dimensional conformation of the docking of ACE inhibitory peptide GFPGLP with DPP-IV inhibitory activity and DPP-IV.

[0039] Figure 10 This is a two-dimensional schematic diagram of the docking interaction force between GFPGLP, an ACE inhibitory peptide with DPP-IV inhibitory activity, and DPP-IV. DETAILED DESCRIPTION

[0040] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0041] 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 this field can implement it. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0042] An embodiment of the present invention provides an ACE inhibitory peptide having DPP-IV inhibitory activity, the amino acid sequence of which is shown in SEQ ID NO.1.

[0043] The present invention also provides a method for preparing the ACE inhibitory peptide, comprising the following steps:

[0044] (1) Enzymatically hydrolyzing the sea cucumber intestines and eggs using papain and trypsin to obtain a sea cucumber intestines and eggs enzymatic hydrolysis product;

[0045] (2) After the sea cucumber intestinal egg enzymatic hydrolysis product is subjected to ultrafiltration and desalting treatment, the sequence of the peptide fragment in the sea cucumber intestinal egg enzymatic hydrolysis product is identified by LC-MS / MS liquid chromatography-mass spectrometry to obtain an enzymatic hydrolysis solution containing the ACE inhibitory peptide with DPP-IV inhibitory activity;

[0046] (3) Screen out ACE inhibitory peptides with DPP-IV inhibitory activity.

[0047] In some specific embodiments, the enzymatic hydrolysis conditions are: the usage of papain is 1000U / g, the usage of trypsin is 800U / g, the usage 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.

[0048] 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 screening ACE inhibitory peptides with potential DPP-IV inhibitory activity based on the docking energy value and activity score with ACE, and analyzing their secondary mass spectrometry structure to obtain the amino acid sequence and structure of the ACE inhibitory peptide.

[0049] The embodiments of the present invention also provide the use of the ACE inhibitory peptide in the preparation of angiotensin-converting enzyme inhibitors.

[0050] An embodiment of the present invention further provides an angiotensin-converting enzyme inhibitor comprising the ACE inhibitory peptide.

[0051] The embodiments of the present invention also provide the use of the ACE inhibitory peptide in the preparation of antihypertensive drugs.

[0052] An embodiment of the present invention further provides a blood pressure lowering drug comprising the ACE inhibitory peptide.

[0053] The embodiments of the present invention also provide use of the ACE inhibitory peptide in the preparation of a dipeptidyl peptidase IV inhibitor.

[0054] An embodiment of the present invention further provides a dipeptidyl peptidase IV inhibitor comprising the ACE inhibitory peptide.

[0055] The embodiments of the present invention also provide the use of the ACE inhibitory peptide in the preparation of hypoglycemic drugs.

[0056] An embodiment of the present invention further provides a hypoglycemic drug comprising the ACE inhibitory peptide.

[0057] The 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, and can also effectively inhibit the activity of DPP-IV, playing a role in preventing or even alleviating diseases such as hypertension and hyperglycemia. It is of great significance to the practical production and theoretical research of multifunctional active peptides.

[0058] The solution proposed by the present invention is described in detail below through specific embodiments:

[0059] Example 1 Preparation of ACE inhibitory peptides from sea cucumber intestinal eggs

[0060] After removal, washing, and homogenization, sea cucumber viscera and eggs were pre-treated using papain and trypsin. The enzymes were hydrolyzed using a combination of papain and trypsin at a dosage of 1000 U / g and 800 U / g, respectively, with a substrate dosage of 15%. The hydrolysis was performed at 65°C for 48 h, with the pH of the system continuously monitored and adjusted to 7.5. The final product of the hydrolysis was ultrafiltered and desalted using a solid-phase extraction cartridge (SEP-PAK C18, Waters, USA) to prepare a sea cucumber viscera hydrolyzate containing ACE inhibitory peptides. The molecular weights and sequences of the peptides prepared from the hydrolyzed sea cucumber viscera were determined using LC-MS / MS.

[0061] Example 2 Screening and Mining of ACE Inhibitory Peptide GFPGLP

[0062] Based on the molecular weight and sequence of peptides from the enzymatic hydrolysate of sea cucumber intestinal eggs identified by LC-MS / MS, peptide sequences with molecular weights less than 2 kDa were selected to construct a peptide database, totaling 1039 entries. Functional scores of these peptides were predicted using the peptide ranker tool (https: / / crdd.osdd.net / raghava / toxinpred / ), and their predicted activity scores were ranked. Toxicity analysis of these peptides was further performed using the ToxinPred tool (https: / / crdd.osdd.net / raghava / toxinpred / ) to ensure that the subsequently screened peptides were nontoxic. The 50 peptides predicted to be nontoxic and with the top 50 activity scores (all predicted to be nontoxic) were then subjected to molecular modeling docking. The docking energy values ​​were used to screen for potential ACE inhibitory peptides.

[0063] The ACE inhibitory peptides with potential functional activity were further docked with ACE by molecular simulation. The three-dimensional conformational diagram 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 ACE inhibitory peptide to dock with ACE was analyzed. The ACE inhibitory peptides with the smallest required energy value and the highest functional score 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 active peptide obtained by screening shows that the amino acid sequence of the active peptide is SEQ ID NO.1: GFPGLP.

[0064] Example 3 Analysis of the binding interaction between ACE inhibitory peptide GFPGLP and ACE

[0065] Based on the molecular simulation docking results during the screening process, the binding sites and force types between the screened ACE inhibitory peptide GFPGLP and ACE were further analyzed to analyze the potential mechanism of action when it exerts its ACE inhibitory activity.

[0066] The results are as follows Figure 2 As shown, the ACE inhibitory peptide GFPGLP can tightly bind 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, mainly Lys368, Asp377, Tyr523, Val518, Glu162, His383, His387, Glu384, Gln281, Ala354, His513, and His353.

[0067] 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.

[0068] 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.

[0069] Example 4 Verification of ACE Inhibitory Activity of ACE Inhibitory Peptide GFPGLP

[0070] The ACE inhibitory activity of the ACE-inhibitory peptide GFPGLP was measured using a visible spectrophotometer in a 96-well plate. 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 the buffer was adjusted to pH 8.3 with NaOH solution and the volume was made up to 100 mL for later use). The assay steps are as follows: different concentrations of ACE inhibitory peptide GFPGLP samples, as well as FAPPG substrate, ACE and buffer were sequentially added to a 96-well plate; the absorbance of the sample system at 340 nm was measured using a microplate reader and recorded; the sample system was placed in a 37°C shaker for incubation for 30 minutes, and the absorbance of the sample system after the reaction was again measured at 340 nm using a microplate reader; the ACE inhibition rate of the sample was determined by calculating the change in absorbance before and after the reaction.

[0071] The calculation formula is:

[0072] ACE inhibition rate of sample % = (1-change in sample absorbance) / change in blank absorbance * 100%.

[0073] The results are as follows Figure 4As 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 assays, the ACE inhibitory peptide GFPGLP can completely inhibit ACE when its concentration is greater than 100 μg / mL. These results indicate that the ACE inhibitory peptide GFPGLP has significant ACE inhibitory activity.

[0074] Example 5 Study on the ACE Inhibitory Mode of ACE Inhibitory Peptide GFPGLP

[0075] The ACE inhibition pattern of the ACE inhibitory peptide GFPGLP was analyzed by measuring the ACE enzyme inhibition kinetics of samples of the ACE inhibitory peptide GFPGLP at different substrate concentrations. The ACE inhibitory peptide GFPGLP samples were diluted to 200 and 400 μg / mL for use, and the substrate FAPPG was diluted to 0.5, 1, and 1.5 mM for use. Reaction systems were prepared using different concentrations of the ACE inhibitory peptide GFPGLP and different concentrations of the FAPPG substrate, and the ACE inhibition rates were determined. The ACE inhibition kinetics were analyzed using the enzyme double reciprocal plotting method and the Michaelis equation, further analyzing the ACE inhibition pattern of the ACE inhibitory peptide GFPGLP.

[0076] 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 velocity V max Unchanged, K m The value increased, indicating that the ACE inhibitory peptide GFPGLP was in a competitive inhibition mode.

[0077] Example 6 Gastrointestinal digestive enzyme stability of ACE inhibitory peptide GFPGLP

[0078] The ACE inhibitory peptide GFPGLP sample was diluted to its IC for ACE inhibition. 50The pH of the system was adjusted to 2 with a 1M HCl solution, and 2% pepsin was added to prepare simulated gastric digestive fluid. A sample of the ACE-inhibiting peptide GFPGLP 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. The treatment lasted for 3 hours to investigate the stability of the ACE-inhibiting peptide GFPGLP in gastric digestive fluid. The enzymatic hydrolysis product was then inactivated by adjusting the pH to 7.5 with 1M NaOH. The resulting gastric hydrolyzate was then treated with trypsin for 3 hours. After the start of the trypsin treatment, samples were taken every 30 minutes and inactivated at 95°C for 10 minutes. The samples were then cooled to 37°C and the in vitro ACE inhibition rate was determined to analyze their stability in intestinal digestive fluid.

[0079] The results are as follows Figure 6 As shown, the concentration of ACE inhibitory peptide GFPGLP is its IC for ACE inhibition 50 At the same concentration, after the above-mentioned gastric digestive juice treatment, 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 ).

[0080] Example 7 Verification of DPP-IV Inhibitory Activity of ACE Inhibitory Peptide GFPGLP

[0081] The DPP-IV inhibitory activity of the ACE-inhibitory peptide GFPGLP was determined using a 96-well plate. The DPP-IV inhibition rate was determined using 1.6 mM Gly-Pro-pNA as a substrate, 0.1 U / mL angiotensin-converting enzyme (ACE), and Tris-HCl as a buffer (100 mM, pH 8.0). The assay steps were as follows: ACE-inhibitory peptide GFPGLP samples at varying concentrations, along with the Gly-Pro-pNA substrate, DPP-IV, and buffer, were sequentially added to a 96-well plate. The sample system was incubated at 37°C for 60 minutes, and the reaction was terminated. The absorbance at 504 nm was measured using a microplate reader and recorded. The DPP-IV inhibition rate was calculated according to the formula.

[0082] The calculation formula is:

[0083] ;

[0084] 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.

[0085] 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 ACE inhibitory peptide GFPGLP on DPP-IV is 89.56%; when the concentration of the ACE inhibitory peptide GFPGLP is 400 μg / mL, the inhibition rate of the ACE inhibitory peptide GFPGLP on 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 DPP-IV inhibitory activity assay results, the ACE inhibitory peptide GFPGLP has significant DPP-IV inhibitory activity.

[0086] Example 8 Molecular simulation docking of ACE inhibitory peptide GFPGLP and DPP-IV

[0087] The ACE inhibitory peptide GFPGLP was docked with DPP-IV through molecular modeling to further determine the mechanism of action of this active peptide in exerting its DPP-IV inhibitory activity. A three-dimensional conformational map of the ACE inhibitory peptide GFPGLP was created using Discovery Studio software, and molecular modeling docking was performed with the catalytic active center of DPP-IV (PDB: 2QT9). Based on the docking results, the interaction forces between the ACE inhibitory peptide GFPGLP and the key amino acid residues in the DPP-IV active center were analyzed.

[0088] The results are as follows Figure 9 As shown, the ACE inhibitory peptide GFPGLP can tightly bind to the DPP-IV active center 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 DPP-IV active center, with the main amino acid residues being Arg471, Arg125, His126, Ser209, Glu205, Glu206, Tyr666, Phe357, Arg358, Val207, Arg669, and Leu57.

[0089] The results of the interaction force analysis between the ACE inhibitory peptide GFPGLP and the amino acid residues of the DPP-IV active center are shown in Figure 2. Figure 10 As shown in Figure 3, the ACE inhibitory peptide GFPGLP forms three hydrogen bonds, seven van der Waals forces, three salt bridges and mutual attraction, and one Pi-Pi stacking force with DPP-IV.

[0090] In summary, the ACE inhibitory peptide GFPGLP can bind to DPP-IV and inhibit DPP-IV activity by interacting with DPP-IV active center residues primarily through hydrogen bonds, van der Waals forces, and salt bridges. This further demonstrates from the perspective of molecular modeling and docking that the ACE inhibitory peptide GFPGLP has significant DPP-IV inhibitory activity.

[0091] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0092] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages or disadvantages of the embodiments.

[0093] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. 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 may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, and all of these should be included in the scope of the claims of the present invention.

Claims

1. An 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, wherein: 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 ACE inhibitory peptide with DPP-IV inhibitory activity; (3) Screening for ACE inhibitory peptides with DPP-IV inhibitory activity; Wherein, in step (1), the enzymatic hydrolysis conditions are as follows: the use amount of papain is 1000 U / g, the use amount of trypsin is 800 U / g, the use amount of sea cucumber intestines and eggs is 15%, the enzymatic hydrolysis is carried out at 65°C for 48 h, and the enzymatic hydrolysis pH is 7.5; In step (3), the screening is specifically as follows: screening out peptide sequences with a molecular weight of less than 2 kDa from an enzymatic hydrolyzate containing the 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. Their secondary mass spectrometry structures were analyzed to obtain the amino acid sequence and structure of the ACE inhibitory peptides.

3. Use of the ACE inhibitory peptide according to claim 1 in any of the following: 1) Application in the preparation of antihypertensive drugs; 2) Application in the preparation of hypoglycemic drugs.

4. A DPP-IV inhibitory drug, characterized in that Comprising the ACE inhibitory peptide according to claim 1.

5. An ACE inhibitory drug, characterized in that: Comprising the ACE inhibitory peptide according to claim 1.

6. A drug for simultaneously inhibiting DPP-IV and ACE, characterized in that: Comprising the ACE inhibitory peptide according to claim 1.

7. A blood pressure lowering drug, characterized in that: Comprising the ACE inhibitory peptide according to claim 1.

8. A hypoglycemic drug, characterized in that: Comprising the ACE inhibitory peptide according to claim 1.

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