Sea cucumber protein heptapeptide with functions of reducing blood pressure and blood sugar and application of sea cucumber protein heptapeptide

Abstract

The invention discloses a sea cucumber protein heptapeptide with functions of reducing blood pressure and blood sugar and application of the sea cucumber protein heptapeptide, and belongs to the technical field of bioactive peptide biology. The amino acid sequence of the sea cucumber protein heptapeptide is LPPGPFP. The sea cucumber protein heptapeptide can significantly inhibit the activity of ACE through a competitive inhibition mode, tolerates the gastrointestinal tract digestion environment, also has DPP-IV inhibition activity, can relieve hypertension and type 2 diabetes mellitus at the same time, and has an application value in preparation of products with the efficacy of reducing blood pressure and blood sugar due to multiple functions of the sea cucumber protein heptapeptide. The sea cucumber protein heptapeptide provided by the invention can significantly inhibit the activity of ACE through a competitive inhibition mode, has DPP-IV inhibition activity, has the characteristics of simple structure, safety, strong activity and the like, and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of active peptide biotechnology, and particularly relates to a sea cucumber protein heptapeptide with the function of lowering blood pressure and blood sugar and an application thereof. Background Art

[0002] Hypertension and type 2 diabetes mellitus (T2DM) are a pair of closely linked chronic health threats that are posing a severe challenge to global public health. The characteristic hyperglycemia state of diabetes is often accompanied by the appearance of hypertension. The two share complex pathophysiological mechanisms such as inflammation, oxidative stress, and impaired endothelial function, which together promote the progression of the disease and increase the risk of cardiovascular complications. The fact that the incidence of cardiovascular disease in diabetic patients is much higher than that in the general population highlights the importance of finding new treatment strategies.

[0003] Traditionally, drug combinations have been widely used to control blood pressure and blood sugar levels, but these therapies may have side effects and often lack comprehensive efficacy. Therefore, the development of inhibitors with dual antihypertensive and hypoglycemic effects has become a hot topic in current research. Angiotensin converting enzyme inhibitors (ACE inhibitors) act on the renin-angiotensin system to promote vasodilation and reduce vascular resistance, thereby achieving a blood pressure-lowering effect. DPP-IV inhibitors (dipeptidyl peptidase 4) protect incretin hormones such as glucagon-like peptide-1 and glucose-dependent insulin polypeptide from degradation, thereby enhancing insulin secretion and improving glucose tolerance, thus playing a key role in blood sugar management.

[0004] In this context, searching for natural bioactive peptides with both ACE inhibitory and DPP-IV inhibitory activities has become a promising research direction. As a marine resource rich in bioactive ingredients, sea cucumbers are often regarded as processing waste, but they are actually rich in proteins and peptides and have great development potential. Studies have shown that functional peptides extracted from sea cucumbers can release components with anti-diabetic effects during gastrointestinal digestion, and some peptides show significant DPP-IV inhibitory activity. In addition, sea cucumber paramyosin has also been found to contain peptides with both ACE inhibitory and DPP-IV inhibitory activities. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a sea cucumber protein heptapeptide having the function of lowering blood pressure and blood sugar and its application.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is a sea cucumber protein heptapeptide with the function of lowering blood pressure and blood sugar, and its amino acid sequence is LPPGPFP (SEQ ID NO.1).

[0008] The second technical solution of the present invention is the use of the sea cucumber protein heptapeptide in the preparation of angiotensin converting enzyme inhibitors.

[0009] The third technical solution of the present invention is an angiotensin converting enzyme inhibitor, comprising the sea cucumber protein heptapeptide.

[0010] A fourth technical solution of the present invention is the use of the sea cucumber protein heptapeptide in the preparation of blood pressure lowering drugs.

[0011] The fifth technical solution of the present invention is a blood pressure lowering drug, comprising the sea cucumber protein heptapeptide.

[0012] The sixth technical solution of the present invention is the use of the sea cucumber protein heptapeptide in the preparation of dipeptidyl peptidase IV inhibitors.

[0013] The seventh technical solution of the present invention is a dipeptidyl peptidase IV inhibitor, comprising the sea cucumber protein heptapeptide.

[0014] The eighth technical solution of the present invention is the use of the sea cucumber protein heptapeptide in the preparation of hypoglycemic drugs.

[0015] The ninth technical solution of the present invention is a blood sugar lowering drug, comprising the sea cucumber protein heptapeptide.

[0016] The beneficial effects of the present invention compared with the prior art are as follows:

[0017] The present invention prepares and identifies a sea cucumber protein heptapeptide with blood pressure and blood sugar lowering functions from sea cucumber viscera. The heptapeptide has ACE and DPP-IV inhibitory activity, 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 preparing products with blood pressure and blood sugar lowering effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The amino acid structure and sequence of sea cucumber protein heptapeptide are shown in the secondary mass spectrometry identification diagram;

[0019] Figure 2 The schematic diagram of the three-dimensional conformation of the docking of sea cucumber protein heptapeptide LPPGPFP and ACE;

[0020] Figure 3 It is a two-dimensional schematic diagram of the docking interaction between sea cucumber protein heptapeptide LPPGPFP and ACE;

[0021] Figure 4 This is the inhibition rate diagram of sea cucumber protein heptapeptide LPPGPFP on ACE at different concentrations;

[0022] Figure 5 This is the ACE inhibition pattern diagram of sea cucumber protein heptapeptide LPPGPFP;

[0023] Figure 6 This is a schematic diagram of the gastric digestion stability of sea cucumber protein heptapeptide LPPGPFP;

[0024] Figure 7 This is a schematic diagram of the intestinal digestion stability of sea cucumber protein heptapeptide LPPGPFP;

[0025] Figure 8 This is a graph showing the inhibition rate of sea cucumber protein heptapeptide LPPGPFP on DPP-IV at different concentrations;

[0026] Fig. 9 The schematic diagram of the three-dimensional conformation of the docking of sea cucumber protein heptapeptide LPPGPFP and DPP-IV;

[0027] Fig.10 This is a two-dimensional schematic diagram of the docking force between the sea cucumber protein heptapeptide LPPGPFP and DPP-IV. DETAILED DESCRIPTION

[0028] 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.

[0029] 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.

[0030] The embodiment of the present invention provides a sea cucumber protein heptapeptide with the function of lowering blood pressure and blood sugar, the amino acid sequence of which is LPPGPFP, and the protein heptapeptide has ACE and DPP-IV inhibitory activity.

[0031] The present invention also provides a method for preparing the sea cucumber protein heptapeptide having the function of lowering blood pressure and blood sugar, comprising the following steps:

[0032] (1) enzymolyzing the sea cucumber viscera with papain, trypsin and flavor protease to obtain a sea cucumber viscera enzymolysis product;

[0033] (2) After the sea cucumber viscera enzymatic hydrolysate is subjected to ultrafiltration and desalting treatment, the peptide sequence in the sea cucumber viscera enzymatic hydrolysate is identified by LC-MS / MS liquid phase mass spectrometry to obtain an enzymatic hydrolysate containing the sea cucumber protein heptapeptide having ACE and DPP-IV inhibitory activity;

[0034] (3) Screen out sea cucumber protein heptapeptides with ACE and DPP-IV inhibitory activity.

[0035] In some specific embodiments, the enzymatic hydrolysis conditions are: the usage amount of papain is 5000U / g, the usage amount of trypsin is 1000U / g, the usage amount of flavor protease is 800U / g, the usage amount of sea cucumber viscera substrate is 10%, firstly, papain and trypsin are mixed for enzymatic hydrolysis at 50°C for 18h, and then flavor protease is added to continue enzymatic hydrolysis at 60°C for 12h, the enzymatic hydrolysis process is continuously stirred, and the enzymatic hydrolysis pH is maintained at 7.5.

[0036] In some specific embodiments, the screening method is: identifying the molecular weight and sequence of peptides in the sea cucumber viscera hydrolysate, screening the peptide sequence composition in the hydrolysate, further predicting the ACE inhibitory activity and DPP-IV inhibitory activity of the peptide sequence, and screening the sea cucumber protein peptides with potential ACE and DPP-IV inhibitory activity based on the docking energy value and activity score, and analyzing its secondary mass spectrometry structure to obtain the amino acid sequence and structure of the sea cucumber protein peptide, and after activity verification, determining it to be a sea cucumber protein heptapeptide with ACE and DPP-IV inhibitory activity.

[0037] The embodiment of the present invention also provides the use of the sea cucumber protein heptapeptide in the preparation of angiotensin converting enzyme inhibitors.

[0038] The embodiment of the present invention further provides an angiotensin converting enzyme inhibitor, comprising the sea cucumber protein heptapeptide.

[0039] The embodiment of the present invention also provides the use of the sea cucumber protein heptapeptide in the preparation of blood pressure lowering drugs.

[0040] The embodiment of the present invention also provides a blood pressure lowering drug, comprising the sea cucumber protein heptapeptide.

[0041] The embodiment of the present invention also provides the use of the sea cucumber protein heptapeptide in the preparation of a dipeptidyl peptidase IV inhibitor.

[0042] The embodiment of the present invention also provides a dipeptidyl peptidase IV inhibitor, comprising the sea cucumber protein heptapeptide.

[0043] The embodiment of the present invention also provides the use of the sea cucumber protein heptapeptide in the preparation of hypoglycemic drugs.

[0044] The embodiment of the present invention also provides a blood sugar lowering drug, comprising the sea cucumber protein heptapeptide.

[0045] The sea cucumber protein heptapeptide with ACE and DPP-IV inhibitory activity provided by the present invention can significantly inhibit the activity of ACE through a competitive inhibition mode, 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, and is of great significance to the actual production and theoretical research of multifunctional active peptides.

[0046] Example 1: Preparation of sea cucumber protein peptides from sea cucumber viscera

[0047] After the sea cucumber viscera was removed, cleaned and homogenized, papain, trypsin and flavor protease were used for composite enzymolysis, wherein the amount of papain used was 5000U / g, the amount of trypsin used was 1000U / g, the amount of flavor protease used was 800U / g, and the amount of sea cucumber viscera substrate was 10%. Papain and trypsin were mixed for enzymolysis at 50°C for 18h, and then flavor protease was added to continue enzymolysis at 60°C for 12h. The enzymolysis process was continuously stirred, and the enzymolysis pH was maintained at 7.5. 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 sea cucumber protein peptides. LC-MS / MS liquid phase mass spectrometry was further used to determine the molecular weight and sequence of each protein peptide prepared in the sea cucumber viscera hydrolyzate.

[0048] Example 2: Screening and mining of sea cucumber protein heptapeptide LPPGPFP

[0049] The molecular weight and sequence of peptides in the enzymatic hydrolysates of sea cucumber viscera were identified by LC-MS / MS liquid chromatography-mass spectrometry, and the ACE inhibitory activity and other functional potential of the peptides were evaluated and predicted using the PeptideRanker functional activity predictor and the Toixinpred safety and toxicity evaluation predictor. Based on the functional scores, sea cucumber protein peptides with potential functional activity were screened.

[0050] The screened sea cucumber protein peptides with potential functional activity were further docked with ACE by molecular simulation, and the three-dimensional conformational diagram of the protein 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 protein peptide to dock with ACE was analyzed, and the protein 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 sea cucumber protein peptide. The results are as follows Figure 1 As shown, the secondary mass spectrum of the screened protein peptide shows that the sea cucumber protein peptide is a heptapeptide, and the amino acid sequence and arrangement are LPPGPFP.

[0051] Example 3: Analysis of the binding interaction between sea cucumber protein heptapeptide LPPGPFP and ACE

[0052] Based on the molecular simulation docking results in the screening process, the binding site and force type between the screened sea cucumber protein heptapeptide LPPGPFP and ACE were further analyzed to analyze its potential mechanism of action when exerting its ACE inhibitory activity.

[0053] The results are as follows Figure 2 As shown in the figure, the sea cucumber protein heptapeptide LPPGPFP can bind tightly to the active center of ACE and can be simulated and docked within the normal analysis time. The sea cucumber protein heptapeptide LPPGPFP interacts with multiple amino acid residues in the active center of ACE, and the main amino acid residues are Phe391 Glu384Trp357 Ser355 His383 Val379 Ala354Val380 Glu376 His353 Val518 Arg522 Tyr523Phe457 Gln281 Thr282.

[0054] The results of the interaction analysis between sea cucumber protein heptapeptide LPPGPFP and the amino acid residues in the active center of ACE are shown in Figure 3 As shown. The sea cucumber protein heptapeptide LPPGPFP forms 4 hydrogen bonds, 1 salt bridge, 1 Pi-Anion force, 3 carbon-hydrogen bonds, 1 Pi-Pi stacking force, 6 alkyl mutual or Pi-alkyl interaction forces, and Zn 2+ Receptor.

[0055] In summary, the sea cucumber protein heptapeptide LPPGPFP 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.

[0056] Example 4: Verification of ACE inhibitory activity of sea cucumber protein heptapeptide LPPGPFP

[0057] The ACE inhibitory activity of sea cucumber protein heptapeptide LPPGPFP 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: different concentrations of sea cucumber protein heptapeptide LPPGPFP, as well as FAPPG substrate, ACE and buffer are added in sequence to a 96-well plate; the absorbance of the sample system at 340 nm is determined using an enzyme marker and recorded; the sample system is placed in a 37°C shaker for shaking and incubation for 30 minutes, and the absorbance of the sample system after the reaction is determined again using an enzyme marker at 340 nm; the ACE inhibition rate of the sample is determined by calculating the change in absorbance before and after the reaction.

[0058] The calculation formula is: ACE inhibition rate of sample % = 1-change in sample absorbance value / change in blank absorbance value.

[0059] The results are as follows Figure 4 As shown, when the concentration of sea cucumber protein heptapeptide LPPGPFP is 10 μg / mL, the inhibition rate of ACE is 10.82%; when the concentration of sea cucumber protein heptapeptide LPPGPFP is 25 μg / mL, the inhibition rate of ACE is 22.62%; when the concentration of sea cucumber protein heptapeptide LPPGPFP is 50 μg / mL, the inhibition rate of ACE is 41.60%; when the concentration of sea cucumber protein heptapeptide LPPGPFP is 100 μg / mL, the inhibition rate of ACE is 87.20%; when the concentration of sea cucumber protein heptapeptide LPPGPFP 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 sea cucumber protein heptapeptide LPPGPFP is greater than 100 μg / mL, it can completely inhibit ACE. The above results show that sea cucumber protein heptapeptide LPPGPFP has significant ACE inhibitory activity.

[0060] Example 5: Determination of ACE inhibition mode of sea cucumber protein heptapeptide LPPGPFP

[0061] The ACE inhibition mode of sea cucumber protein heptapeptide LPPGPFP was analyzed by measuring the ACE enzyme inhibition kinetics of sea cucumber protein heptapeptide LPPGPFP samples with different concentrations under different concentrations of substrates. The sea cucumber protein heptapeptide LPPGPFP 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 sea cucumber protein heptapeptide LPPGPFP 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 sea cucumber protein heptapeptide LPPGPFP was analyzed.

[0062] The results are as follows Figure 5 As shown in Figure 2, as the concentration of sea cucumber protein heptapeptide LPPGPFP increases, the maximum reaction rate V max No change, K m The value increases, indicating that the sea cucumber protein heptapeptide LPPGPFP is in a competitive inhibition mode.

[0063] Example 6: Stability of sea cucumber protein heptapeptide LPPGPFP to gastrointestinal digestive enzymes

[0064] The sea cucumber protein heptapeptide LPPGPFP sample was diluted to its IC value for ACE inhibition. 50 The value concentration was adjusted to 2 with a 1M HCl solution, and pepsin was added at a concentration of 2% to prepare a simulated gastric digestive fluid. The sea cucumber protein heptapeptide LPPGPFP 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 total treatment was 3 hours to analyze the stability of the gastric digestive fluid. Subsequently, the enzymatic hydrolysis product was inactivated by adjusting the pH to 7.5 with a 1M concentration of NaOH, and the gastric hydrolyzate was obtained for trypsin treatment for 3 hours. After the trypsin treatment began, 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 intestinal digestion stability.

[0065] The results are as follows Figure 6 As shown, the sea cucumber protein heptapeptide LPPGPFP is its IC for ACE inhibition 50 At 1.544 μg / mL, after being treated with the above-mentioned gastric digestive juice, the inhibition rate of the digested sea cucumber protein heptapeptide LPPGPFP on ACE changed from 50.08% to 42.38%, with a small decrease in inhibition rate, and still having good ACE inhibitory activity, indicating that the sea cucumber protein heptapeptide LPPGPFP is tolerant to gastric digestive juice. After being treated with intestinal digestive juice for 3 hours, its inhibition rate on ACE finally changed to 40.96%, indicating that the sea cucumber protein heptapeptide LPPGPFP is tolerant to intestinal digestive juice (see Figure 7 ).

[0066] Example 7: Verification of DPP-IV inhibitory activity of sea cucumber protein heptapeptide LPPGPFP

[0067] The DPP-IV inhibitory activity of sea cucumber protein heptapeptide LPPGPFP 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 DPP-IV; Tris-HCl as buffer matrix (100mM, pH 8.0). The determination steps are: different concentrations of sea cucumber protein heptapeptide LPPGPFP 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 instrument and recorded, and the determined DPP-IV inhibition rate was obtained according to the calculation formula.

[0068] The calculation formula is:

[0069] 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.

[0070] The results are as follows Figure 8 As shown, the concentration of sea cucumber protein heptapeptide LPPGPFP is between 100 μg / mL and 800 μg / mL, which can inhibit DPP-IV, and the inhibition rate can reach 69.89%, indicating that sea cucumber protein heptapeptide LPPGPFP has effective DPP-IV inhibitory activity.

[0071] Example 8: Molecular simulation docking of sea cucumber protein heptapeptide LPPGPFP and DPP-IV

[0072] The sea cucumber protein heptapeptide LPPGPFP was docked with DPP-IV by molecular simulation to further determine the mechanism of action of the sea cucumber protein peptide when it exerts its DPP-IV inhibitory activity. The three-dimensional conformational diagram of the sea cucumber protein heptapeptide LPPGPFP 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 sea cucumber protein heptapeptide LPPGPFP and the key amino acid residues in the active center of DPP-IV was analyzed based on the docking results.

[0073] The results are as follows Fig. 9The sea cucumber protein heptapeptide LPPGPFP can be tightly bound to the DPP-IV active center and can be simulated and docked within the normal analysis time. The live sea cucumber protein heptapeptide LPPGPFP interacts with multiple amino acid residues in the DPP-IV active center, and the main amino acid residues are Ser209, Ala210, Phe208, Glu361, Arg358, Ser458, Val459, Ser460, Arg471, Glu408, Tyr456, Ile407, Ile418, and Gly406.

[0074] The results of the interaction analysis between the sea cucumber protein heptapeptide LPPGPFP and the amino acid residues in the active center of DPP-IV are shown in Fig.10 As shown in the figure, the sea cucumber protein heptapeptide LPPGPFP forms one hydrogen bond, 11 Wandwal forces, one salt bridge, one mutual attraction, and one alkyl interaction with DPP-IV.

[0075] In summary, the sea cucumber protein heptapeptide LPPGPFP can bind to DPP-IV, and mainly interacts with the DPP-IV active center residues through hydrogen bonds, Van der Waals forces, salt bridges and alkyl interactions to inhibit the activity of DPP-IV. That is, from the perspective of molecular simulation docking, it is further proved that the sea cucumber protein heptapeptide LPPGPFP has DPP-IV inhibitory activity.

Claims

1. A sea cucumber protein heptapeptide with the function of lowering blood pressure and blood sugar, characterized in that: The amino acid sequence of the sea cucumber protein heptapeptide is LPPGPFP.

2. The use of the sea cucumber protein heptapeptide according to claim 1, characterized in that: The application is to use the sea cucumber protein heptapeptide according to claim 1 to prepare angiotensin converting enzyme inhibitors, antihypertensive drugs, dipeptidyl peptidase IV inhibitors or blood sugar lowering drugs.

3. An angiotensin converting enzyme inhibitor, characterized in that The angiotensin converting enzyme inhibitor comprises the sea cucumber protein heptapeptide according to claim 1.

4. A blood pressure lowering drug, characterized in that: The blood pressure lowering drug comprises the sea cucumber protein heptapeptide according to claim 1.

5. A dipeptidyl peptidase IV inhibitor, characterized in that: The invention comprises the sea cucumber protein heptapeptide as claimed in claim 1.

6. A hypoglycemic drug, characterized in that: The blood sugar lowering drug comprises the sea cucumber protein heptapeptide according to claim 1.

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