A sea cucumber protein heptapeptide with blood pressure and blood sugar lowering functions and its application

Abstract

The present invention discloses a sea cucumber protein heptapeptide with blood pressure and blood sugar lowering functions and its application, belonging to the field of active peptide biotechnology. 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, tolerate the gastrointestinal digestion environment, and also has DPP-IV inhibitory activity, playing a role in alleviating both hypertension and type 2 diabetes. Its multiple functions make it have application value in the preparation of products with blood pressure and blood sugar lowering effects. The sea cucumber protein heptapeptide involved in the present invention can significantly inhibit the activity of ACE through a competitive inhibition mode, and at the same time has DPP-IV inhibitory activity. It has the characteristics of simple structure, safety, strong activity, etc., and has broad application prospects.

Description

Technical Field

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

[0002] Hypertension and type 2 diabetes mellitus (T2DM), as a pair of closely related chronic health threats, are posing a severe challenge to global public health. The characteristic hyperglycemic state of diabetes is often accompanied by the emergence of hypertension. Both share complex pathophysiological mechanisms such as inflammation, oxidative stress, and impaired endothelial function. These mechanisms jointly drive the progression of the diseases and increase the risk of cardiovascular complications. The incidence of cardiovascular diseases in diabetic patients is much higher than that in the general population, which 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 cause side effects and often lack comprehensive efficacy. Therefore, the development of inhibitors with dual blood pressure and blood sugar lowering effects has become a current research hotspot. Angiotensin converting enzyme inhibitors (ACE inhibitors) act on the renin-angiotensin system to promote vasodilation and reduce vascular resistance, thereby achieving the blood pressure lowering effect. DPP-IV inhibitors (dipeptidyl peptidase 4) play a key role in blood sugar management by protecting incretin hormones such as glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide from degradation, thereby enhancing insulin secretion and improving glucose tolerance.

[0004] In this context, finding natural bioactive peptides with both ACE inhibitory and DPP-IV inhibitory activities has become a promising research direction. Sea cucumbers, as a marine resource rich in bioactive components, although their visceral parts are often regarded as processing wastes, are actually rich in proteins and polypeptides and have great development potential. Research shows that functional peptides extracted from sea cucumbers can release components with antidiabetic effects during gastrointestinal digestion, and some peptide segments exhibit significant DPP-IV inhibitory activity. In addition, sea cucumber paramyosin has also been found to contain peptide substances 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 with blood pressure and blood sugar lowering functions 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 blood pressure and blood sugar lowering functions, and its amino acid sequence is LPPGPFP (SEQ ID NO.1).

[0008] Another technical solution of the present invention is the application of the sea cucumber protein heptapeptide in the preparation of an angiotensin-converting enzyme inhibitor.

[0009] Another technical solution of the present invention is an angiotensin-converting enzyme inhibitor containing the sea cucumber protein heptapeptide.

[0010] Another technical solution of the present invention is the application of the sea cucumber protein heptapeptide in the preparation of a blood pressure lowering drug.

[0011] Another technical solution of the present invention is a blood pressure lowering drug containing the sea cucumber protein heptapeptide.

[0012] Another technical solution of the present invention is the application of the sea cucumber protein heptapeptide in the preparation of a dipeptidyl peptidase IV inhibitor.

[0013] Another technical solution of the present invention is a dipeptidyl peptidase IV inhibitor containing the sea cucumber protein heptapeptide.

[0014] Another technical solution of the present invention is the application of the sea cucumber protein heptapeptide in the preparation of a blood sugar lowering drug.

[0015] Another technical solution of the present invention is a blood sugar lowering drug containing the sea cucumber protein heptapeptide.

[0016] Advantages of the present invention compared with the prior art:

[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 activities, can significantly inhibit the activity of ACE through a competitive inhibition mode, has gastrointestinal digestion tolerance, and also has DPP-IV inhibitory activity, which can relieve hypertension and type 2 diabetes and their related complications, and has application value in the preparation of products with blood pressure and blood sugar lowering effects. Description of the Drawings

[0018] Figure 1 It is the amino acid structure and sequence secondary mass spectrometry identification diagram of the sea cucumber protein heptapeptide;

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

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

[0021] Figure 4 Inhibitory rate graph of sea cucumber protein heptapeptide LPPGPFP against ACE at different concentrations;

[0022] Figure 5 Inhibitory mode graph of sea cucumber protein heptapeptide LPPGPFP against ACE;

[0023] Figure 6 Gastric juice digestion stability schematic diagram of sea cucumber protein heptapeptide LPPGPFP;

[0024] Figure 7 Intestinal digestion stability schematic diagram of sea cucumber protein heptapeptide LPPGPFP;

[0025] Figure 8 Inhibitory rate graph of sea cucumber protein heptapeptide LPPGPFP against DPP-IV at different concentrations;

[0026] Figure 9 Three-dimensional docking conformation schematic diagram of sea cucumber protein heptapeptide LPPGPFP and DPP-IV;

[0027] Figure 10 Two-dimensional schematic diagram of the docking interaction force between sea cucumber protein heptapeptide LPPGPFP and DPP-IV. Detailed implementation methods

[0028] The technical solutions of the present invention will be further explained through examples below, but the protection scope of the present invention is not limited by any form of the examples.

[0029] The technical solutions described in the present invention are all conventional solutions in the art unless otherwise specified. The reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

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

[0031] An embodiment of the present invention also provides a preparation method of the sea cucumber protein heptapeptide with blood pressure and blood sugar lowering functions, including the following steps:

[0032] (1) Using papain, trypsin and flavor protease to enzymatically hydrolyze sea cucumber viscera to obtain a sea cucumber viscera enzymatic hydrolysis product;

[0033] (2) After the sea cucumber viscera enzymatic hydrolysis product is subjected to ultrafiltration and desalting treatment, the sequence of the peptide segments in the sea cucumber viscera enzymatic hydrolysis product is identified by LC-MS / MS liquid chromatography-mass spectrometry tandem method to obtain an enzymatic hydrolysis solution containing the sea cucumber protein heptapeptide with ACE and DPP-IV inhibitory activities;

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

[0035] In some specific embodiments, the enzymatic hydrolysis conditions are as follows: the dosage of papain is 5000 U / g, the dosage of trypsin is 1000 U / g, the dosage of flavor protease is 800 U / g, the dosage of sea cucumber viscera substrate is 10%, first use the mixed enzymes of papain and trypsin to hydrolyze at 50 °C for 18 h, then add flavor protease and continue to hydrolyze at 60 °C for 12 h. Stir constantly during the enzymatic hydrolysis process, and keep the enzymatic hydrolysis pH at 7.5.

[0036] In some specific embodiments, the screening method is as follows: identify the molecular weight and sequence of the peptide segments in the enzymatic hydrolysis product of sea cucumber viscera, screen out the peptide segment sequence composition in the enzymatic hydrolysis product, further predict the ACE inhibitory activity and DPP-IV inhibitory activity of the peptide segment sequence, and based on the docking energy value and activity score, screen out the sea cucumber protein peptides with potential ACE and DPP-IV inhibitory activities, and analyze the secondary mass spectrometry structure thereof to obtain the amino acid sequence and structure of the sea cucumber protein peptide. After activity verification, determine it as the sea cucumber protein heptapeptide with ACE and DPP-IV inhibitory activities.

[0037] The embodiment of the present invention also provides the application of the sea cucumber protein heptapeptide in the preparation of an angiotensin-converting enzyme inhibitor.

[0038] The embodiment of the present invention also provides an angiotensin-converting enzyme inhibitor containing the sea cucumber protein heptapeptide.

[0039] The embodiment of the present invention also provides the application of the sea cucumber protein heptapeptide in the preparation of a hypotensive drug.

[0040] The embodiment of the present invention also provides a hypotensive drug containing the sea cucumber protein heptapeptide.

[0041] The embodiment of the present invention also provides the application 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 containing the sea cucumber protein heptapeptide.

[0043] The embodiment of the present invention also provides the application of the sea cucumber protein heptapeptide in the preparation of an antidiabetic drug.

[0044] The embodiment of the present invention also provides an antidiabetic drug containing the sea cucumber protein heptapeptide.

[0045] The sea cucumber protein heptapeptide with ACE and DPP-IV inhibitory activities provided by the present invention can significantly inhibit the activity of ACE through a competitive inhibition mode, 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.

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

[0047] After pretreatment of removing, cleaning and homogenizing the sea cucumber viscera, papain, trypsin and flavor protease were used for complex enzymatic hydrolysis. The dosage of papain was 5000 U / g, the dosage of trypsin was 1000 U / g, the dosage of flavor protease was 800 U / g, and the dosage of sea cucumber viscera substrate was 10%. First, papain and trypsin were used for mixed enzymatic hydrolysis at 50 °C for 18 h, then flavor protease was added and the enzymatic hydrolysis was continued at 60 °C for 12 h. The enzymatic hydrolysis process was continuously stirred, and the pH of the enzymatic hydrolysis was maintained at 7.5. The final product of the enzymatic hydrolysis of the sea cucumber viscera was subjected to ultrafiltration treatment and desalting treatment using a solid-phase extraction column (SEP-PAK C18 solid-phase extraction column, Waters, USA) to prepare a sea cucumber viscera hydrolysate containing sea cucumber protein peptides. Further, LC-MS / MS liquid chromatography-mass spectrometry was used to determine the molecular weights and sequences of the various protein peptide segments prepared in the enzymatic hydrolysate of the sea cucumber viscera.

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

[0049] According to the molecular weights and sequences of the peptide segments in the enzymatic hydrolysate of the sea cucumber viscera identified by LC-MS / MS liquid chromatography-mass spectrometry, the PeptideRanker functional activity predictor and the Toixinpred safety and toxicity evaluation predictor were further used to evaluate and predict the ACE inhibitory activity and other functional potentials of the peptide segments, and based on the functional scores, sea cucumber protein peptides with potential functional activities were screened.

[0050] Furthermore, molecular simulation docking of the screened sea cucumber protein peptides with potential functional activities with ACE was carried out. The Discovery studio software was used to draw the three-dimensional conformation diagram of the protein peptide, and molecular simulation docking was carried out with the catalytic active center of ACE (PDB: 1o8a). Based on the docking results, the energy value required for the docking of the protein peptide with ACE was analyzed, and the protein peptide with a small required energy value and a preferred functional score was selected, and its secondary mass spectrometry structure was analyzed to obtain the amino acid sequence and structure of the sea cucumber protein peptide. The results are as Figure 1 shown. The secondary mass spectrometry diagram 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 the sea cucumber protein heptapeptide LPPGPFP and ACE

[0052] Based on the molecular simulation docking results during the screening process, further analyze the binding sites and types of interactions between the selected sea cucumber protein heptapeptide LPPGPFP and ACE, in order to analyze the potential mechanism of action when it exerts its ACE inhibitory activity.

[0053] The results are as Figure 2 shown. The sea cucumber protein heptapeptide LPPGPFP can tightly bind to the active center of ACE and can perform simulated docking within the normal analysis time. Moreover, 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, Glu384, Trp357, Ser355, His383, Val379, Ala354, Val380, Glu376, His353, Val518, Arg522, Tyr523, Phe457, Gln281, Thr282.

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

[0055] All in all, the sea cucumber protein heptapeptide LPPGPFP can bind to ACE, and mainly interacts with the residues in the active center of ACE through hydrogen bonds, hydrophobic interactions and Zn 2+ binding to inhibit the activity of ACE.

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

[0057] The ACE inhibitory activity of the sea cucumber protein heptapeptide LPPGPFP was determined using a visible spectrophotometer method with a 96-well plate. 1 mM N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine (FAPPG) was used as the substrate; 0.1 U / mL angiotensin-converting enzyme (ACE); HEPES was used as the buffer matrix (weigh 1.901 g of HEPES reagent and 1.755 g of NaCl reagent, dissolve in an appropriate amount of deionized water, and then adjust the pH of the buffer to 8.3 with NaOH solution and make up to 100 mL for standby) to determine the ACE inhibition rate. The determination steps were as follows: Add different concentrations of the sea cucumber protein heptapeptide LPPGPFP, as well as the FAPPG substrate, ACE, and buffer, etc. in sequence to the 96-well plate; Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value of the sample addition system at 340 nm and record it; Place the sample addition system in a shaker incubator at 37 °C and shake and incubate for 30 min, and then use the ELISA reader to measure the absorbance value of the reacted sample addition system at 340 nm again; The ACE inhibition rate of the sample was determined by calculating the change in absorbance value before and after the reaction.

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

[0059] The results were as Figure 4 shown. When the concentration of the sea cucumber protein heptapeptide LPPGPFP was 10 μg / mL, the inhibition rate of ACE was 10.82%; when the concentration of the sea cucumber protein heptapeptide LPPGPFP was 25 μg / mL, the inhibition rate of ACE was 22.62%; when the concentration of the sea cucumber protein heptapeptide LPPGPFP was 50 μg / mL, the inhibition rate of ACE was 41.60%; when the concentration of the sea cucumber protein heptapeptide LPPGPFP was 100 μg / mL, the ACE inhibition rate was 87.20%; when the concentration of the sea cucumber protein heptapeptide LPPGPFP continued to increase, its inhibition rate of ACE could reach 100% (see Figure 4 ). According to the results of the ACE inhibitory activity determination, when the concentration of the sea cucumber protein heptapeptide LPPGPFP was greater than 100 μg / mL, it could completely inhibit ACE. The above results indicate that the sea cucumber protein heptapeptide LPPGPFP has significant ACE inhibitory activity.

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

[0061] The ACE inhibition mode of the sea cucumber protein heptapeptide LPPGPFP was analyzed by measuring the ACE enzyme inhibition kinetics of samples of different concentrations of the sea cucumber protein heptapeptide LPPGPFP under different concentrations of substrates. The samples of the sea cucumber protein heptapeptide LPPGPFP were diluted to 200 and 400 μg / mL for standby, and the substrate FAPPG was diluted to concentrations of 0.5, 1, and 1.5 mM for standby. Reaction systems were prepared by combining different concentrations of the sea cucumber protein heptapeptide LPPGPFP with different concentrations of the FAPPG substrate, and their ACE inhibition rates were measured. According to the double-reciprocal plotting method of the enzyme and the Michaelis equation, the inhibition kinetics of ACE was analyzed, and further the ACE inhibition mode of the sea cucumber protein heptapeptide LPPGPFP was analyzed.

[0062] The results are as Figure 5 shown. As the concentration of the sea cucumber protein heptapeptide LPPGPFP increased, the maximum reaction rate V of the enzyme max remained unchanged, and the K m value increased, indicating that the sea cucumber protein heptapeptide LPPGPFP was in a competitive inhibition mode.

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

[0064] The sample of the sea cucumber protein heptapeptide LPPGPFP was diluted to the concentration of its IC 50 value for ACE inhibition. The pH of the system was adjusted to 2 with a 1 M HCl solution, and pepsin was added at a concentration of 2% to prepare a simulated gastric digestive fluid. The sample of the sea cucumber protein heptapeptide LPPGPFP was mixed with the pepsin solution and incubated at 37 °C. After the reaction, samples were taken every 30 min to measure their ACE inhibition rates, and the treatment lasted for 3 h to analyze the stability against gastric digestive fluid. Subsequently, the enzyme hydrolysis products were inactivated by adjusting the pH to 7.5 with 1 M NaOH, and the gastric hydrolysis products were used for trypsin treatment for 3 h. After the start of trypsin treatment, samples were taken every 30 min, inactivated at 95 °C for 10 min, cooled to 37 °C, and the in vitro ACE inhibition rate was measured to analyze its intestinal digestive stability.

[0065] The results are as Figure 6 shown. When the sea cucumber protein heptapeptide LPPGPFP was at the concentration of its IC 50 value for ACE inhibition, after treatment with the above gastric digestive fluid, the inhibition rate of the digested sea cucumber protein heptapeptide LPPGPFP against ACE changed from 50.08% to 42.38%, and the decrease in the inhibition rate was small, and it still had good ACE inhibition activity, indicating that the sea cucumber protein heptapeptide LPPGPFP had tolerance to gastric digestive fluid. Subsequently, after treatment with intestinal digestive fluid for 3 h, its inhibition rate against ACE finally became 40.96%, indicating that the sea cucumber protein heptapeptide LPPGPFP had tolerance to intestinal digestive fluid (see Figure 7 ).

[0066] Example 7: Verification of the 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. 1.6 mM Gly-Pro-pNA was used as the substrate; 0.1 U / mL DPP-IV; Tris-HCl was used as the buffer matrix (100 mM, pH 8.0) for the determination of the DPP-IV inhibition rate. The determination steps were as follows: Different concentrations of the sea cucumber protein heptapeptide LPPGPFP samples, as well as the Gly-Pro-pNA substrate, DPP-IV, and buffer, were added successively to the 96-well plate; the sample-adding system was incubated at 37 °C for 60 min and the reaction was terminated, and the absorbance value of the sample-adding system at 504 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader and recorded. The DPP-IV inhibition rate was calculated 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 Figure 8 shown. When the concentration of the sea cucumber protein heptapeptide LPPGPFP is between 100 μg / mL and 800 μg / mL, it can inhibit DPP-IV, and the inhibition rate can reach 69.89%, indicating that the 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 and DPP-IV were subjected to molecular simulation docking to further determine the mechanism of action when the sea cucumber protein peptide exerts its DPP-IV inhibitory activity. The Discovery studio software was used to draw the three-dimensional conformation diagram of the sea cucumber protein heptapeptide LPPGPFP, and molecular simulation docking was performed with the catalytic active center of DPP-IV (PDB: 2QT9), and the interaction forces between the key amino acid residues of the sea cucumber protein heptapeptide LPPGPFP and the DPP-IV active center were analyzed based on the docking results.

[0073] The results are as Figure 9The sea cucumber protein heptapeptide LPPGPFP shown can bind tightly to the active center of DPP-IV and can perform simulated docking within the normal analysis time. Moreover, the live sea cucumber protein heptapeptide LPPGPFP interacts with various amino acid residues in the active center of DPP-IV, and the main amino acid residues are Ser209, Ala210, Phe208, Glu361, Arg358, Ser458, Val459, Ser460, Arg471, Glu408, Tyr456, Ile407, Ile418, Gly406.

[0074] The analysis results of the interaction forces between the sea cucumber protein heptapeptide LPPGPFP and the amino acid residues in the active center of DPP-IV are as Figure 10 shown. The sea cucumber protein heptapeptide LPPGPFP forms 1 hydrogen bond, 11 van der Waals forces, 1 salt bridge, 1 mutual attraction, and 1 alkyl interaction with DPP-IV.

[0075] All in all, the sea cucumber protein heptapeptide LPPGPFP can bind to DPP-IV, and mainly interacts with the residues in the active center of DPP-IV through hydrogen bonds, van der Waals forces, salt bridge interactions and alkyl interactions, thereby inhibiting 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 according to claim 1.

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

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