Polypeptide with antihypertensive and hypoglycemic effects as well as preparation method and application thereof

Abstract

The invention discloses a polypeptide with antihypertensive and hypoglycemic effects and a preparation method and application thereof. The invention discloses a polypeptide with effects of reducing blood pressure and reducing blood sugar. The amino acid sequence of the polypeptide is shown as SEQ ID NO.1, or SEQ ID NO.3, or SEQ ID NO.4, or SEQ ID NO.5, or SEQ ID NO.6, or SEQ ID NO.8, or SEQ ID NO.9, or SEQ ID NO.10. The invention further discloses a preparation method of the polypeptide. The application of the polypeptide is used for manufacturing medicines with the functions of reducing blood pressure and blood sugar. The invention also discloses a preparation method of the polypeptide with the effects of reducing blood pressure and blood sugar. The polypeptide provided by the invention not only can effectively inhibit ACE activity and reduce blood pressure, but also can regulate the blood sugar level by inhibiting DPP4 activity, and the preparation method is simple, the production cost is low, and the biological activity is high.

Description

Technical Field

[0001] The present invention relates to the technical field of almond processing, and more specifically to a polypeptide having the effects of lowering blood pressure and blood sugar, and a preparation method and application thereof. Background Art

[0002] Angiotensin-I Converting Enzyme (ACE) is a key enzyme in the renin-angiotensin system that catalyzes the conversion of angiotensin I into angiotensin II, a potent vasoconstrictor that significantly increases blood pressure. ACE is also involved in the degradation of bradykinin, which has a vasodilatory effect. Therefore, the activity of ACE directly affects blood pressure regulation. Inhibiting the activity of ACE is an important way to treat hypertension, and ACE inhibitors are therefore widely used in the development of antihypertensive drugs.

[0003] At present, there are many ACE inhibitors on the market, such as teprotide, etc. These compounds have shown significant ACE inhibitory activity in both in vitro and in vivo experiments and can effectively lower blood pressure. In addition, natural ACE inhibitors based on food-derived peptides have attracted widespread attention due to their fewer side effects and good biocompatibility. Studies have shown that some naturally occurring peptides have strong ACE inhibitory activity and can be used as potential candidates for the development of new antihypertensive drugs.

[0004] Although existing ACE inhibitors such as teprotide have significant effects in lowering blood pressure, research on peptide drugs that have both blood pressure and blood sugar lowering effects is still insufficient. In addition, peptide drugs in the prior art may have problems such as complex preparation process, high production cost, and low bioavailability, which limit their clinical application. Summary of the invention

[0005] To this end, the technical problem to be solved by the present invention is to provide a polypeptide with antihypertensive and hypoglycemic effects, and a preparation method and application thereof. The polypeptide can not only effectively inhibit ACE activity and lower blood pressure, but also regulate blood sugar levels by inhibiting DPP4 activity, and the preparation method is simple, the production cost is low, and the biological activity is high.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] A polypeptide having the effects of lowering blood pressure and blood sugar, wherein the amino acid sequence of the polypeptide is shown in SEQ ID NO.1, or SEQ ID NO.3, or SEQ ID NO.4, or SEQ ID NO.5, or SEQ ID NO.6, or SEQ ID NO.8, or SEQ ID NO.9, or SEQ ID NO.10.

[0008] The above-mentioned polypeptides, the polypeptide with amino acid sequences as shown in SEQ ID NO.1 and the polypeptide with amino acid sequences as shown in SEQ ID NO.8 are both polypeptides having blood pressure lowering effects, and the polypeptide with amino acid sequences as shown in SEQ ID NO.10 is a polypeptide having blood sugar lowering effects.

[0009] The application of the above-mentioned polypeptides is to use any one of the polypeptides for manufacturing a drug with the function of lowering blood pressure and / or blood sugar.

[0010] The above application is to use the polypeptide with an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.8 to manufacture a drug with a blood pressure lowering function, and to use the polypeptide with an amino acid sequence as shown in SEQ ID NO.10 to manufacture a drug with a blood sugar lowering function.

[0011] The method for preparing a polypeptide comprises the following steps:

[0012] S1, debittering, peeling and crushing almonds, and then subcritical defatting to obtain defatted almond powder;

[0013] S2. Using running water to remove impurities from defatted almond flour;

[0014] S3, dissolving the defatted almond powder after impurities removal with alkali, then removing insoluble matter, subjecting the solution to acid precipitation, washing and drying the precipitate obtained by the acid precipitation, and obtaining almond protein powder;

[0015] S4, hydrolyzing the almond protein powder using a protease to obtain an almond protein hydrolyzate;

[0016] S5. Purify and freeze-dry the almond protein hydrolysate to obtain the above-mentioned polypeptide.

[0017] In the above-mentioned preparation method, in step S1, the solvent used in subcritical defatting is butane; in step S2, when removing impurities, the defatted almond powder is ultrafiltered using flowing water; the ultrafiltration membrane used in ultrafiltration is a 3kDA ultrafiltration membrane, the water temperature during ultrafiltration is 4-10°C, and the ultrafiltration time is 22-26 hours. Using butane as a subcritical degreasing solvent can effectively remove fat from almonds, retain protein, and provide an efficient matrix for subsequent hydrolysis. Using a 3kDa ultrafiltration membrane for ultrafiltration at 4-10°C helps to remove impurities, retain target proteins, and prevent protein denaturation.

[0018] In the above preparation method, in step S3, during alkali dissolution, the mass / volume ratio of defatted almond powder to the alkali dissolution reaction system is 1g: (15-20)mL, the pH of the alkali dissolution reaction system is 8.8-9.5, and the alkali dissolution time is 1-3 hours; the acid used during acid precipitation is hydrochloric acid, the concentration of hydrochloric acid is 8-12 mol / L, and the pH of the acid precipitation reaction system is 3.3-4.7. The pH, reaction time and mass / volume ratio of alkali dissolution affect the solubility and structure of the protein, and the concentration of hydrochloric acid and the pH of acid precipitation regulate the precipitation efficiency of the protein and the purity of the product, affecting the purity of the final polypeptide.

[0019] In the above preparation method, in step S4, papain is used to hydrolyze almond protein powder; in the hydrolysis reaction system, the concentration of almond protein powder is 30-50 mg / mL, and the mass ratio of papain to almond protein powder is 18000-20000 U / g; the pH of the hydrolysis reaction system is 6.5-7.2, the temperature of the hydrolysis reaction is 55-65°C, and the hydrolysis reaction time is 3-4 hours. When using protease to hydrolyze protein to prepare polypeptides, the type, dosage and hydrolysis conditions of the enzyme have a significant effect on the product polypeptide sequence. Different enzymes generally have different specificities, and the effect of enzyme dosage will also affect the degree of protein degradation. Higher concentrations of enzymes will accelerate the enzymatic reaction and produce shorter peptides. Hydrolysis conditions such as pH, temperature and time will affect the activity of the enzyme. Under the above conditions, using papain to hydrolyze almond protein powder can produce polypeptides with antihypertensive and hypoglycemic effects.

[0020] In the above-mentioned preparation method, in the hydrolysis reaction system, the concentration of almond protein powder is 40 mg / mL, the mass ratio of papain to almond protein powder is 20000 U / g; the pH of the hydrolysis reaction system is 6.5, the hydrolysis temperature is 60° C., and the hydrolysis time is 220 minutes.

[0021] In the above preparation method, in step S5, when purifying the almond protein hydrolysate, ultrafiltration is used to remove components with a molecular weight less than 3 kDa in the almond protein hydrolysate, and then the remaining components are purified by gel chromatography and reverse phase chromatography in sequence.

[0022] The technical solution of the present invention achieves the following beneficial technical effects:

[0023] 1. The present invention provides polypeptides that have both hypotensive and hypoglycemic effects, and tests the binding ability of these polypeptides with angiotensin-converting enzyme ACE and dipeptidyl peptidase-IV DPP4 through molecular docking experiments. The experimental results show that these polypeptides can bind to the active sites of ACE and DPP4, which indicates that these polypeptides can inhibit the activity of ACE and DPP4 and play the role of lowering blood pressure and blood sugar. The inhibition rate test experiment further proves that YLSF and WNPRDPQ can inhibit the activity of ACE and DPP4, HLPL can inhibit the activity of DPP4, and the binding energy test results show that the binding of these three polypeptides to ACE and DPP4 is relatively stable.

[0024] 2. The polypeptide provided by the present invention can interact with ACE, DPP4 and other enzymes related to regulating blood pressure and blood sugar. The present invention provides a new strategy for developing new drugs for treating diseases such as hypertension and diabetes, and has important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The biological activity prediction results of the BIOPEP-UWM database for the polypeptide sequence YLSF in the embodiment of the present invention;

[0026] Figure 2 Schematic diagram of the conformation of the complex formed after docking of YLSF and ACE in an embodiment of the present invention;

[0027] Figure 3 Schematic diagram of the interaction between YLSF and different amino acid residues of ACE in the embodiment of the present invention;

[0028] Figure 4 Schematic diagram of the conformation of the complex formed after SPHWNVN and ACE are docked in an embodiment of the present invention;

[0029] Figure 5 Schematic diagram of the interaction between SPHWNVN and different amino acid residues of ACE in the embodiment of the present invention;

[0030] Figure 6 Schematic diagram of the conformation of the complex formed after ESWNPRDPQF and ACE are docked in an embodiment of the present invention;

[0031] Figure 7 Schematic diagram of the interaction between ESWNPRDPQF and different amino acid residues of ACE in the embodiment of the present invention;

[0032] Figure 8Schematic diagram of the conformation of the complex formed after GNLYSPHWNVN and ACE are docked in an embodiment of the present invention;

[0033] Fig. 9 Schematic diagram of the interaction between GNLYSPHWNVN and different amino acid residues of ACE in the embodiment of the present invention;

[0034] Fig.10 Schematic diagram of the conformation of the complex formed after HNLPLLR and ACE docking in an embodiment of the present invention;

[0035] Fig.11 Schematic diagram of the interaction between HNLPLLR and different amino acid residues of ACE in an embodiment of the present invention;

[0036] Fig.12 Schematic diagram of the conformation of the complex formed after WNPRDPQ and ACE are docked in an embodiment of the present invention;

[0037] Fig.13 Schematic diagram of the interaction between WNPRDPQ and different amino acid residues of ACE in an embodiment of the present invention;

[0038] Fig.14 Schematic diagram of the conformation of the complex formed after docking of NHLLPLR and ACE in an embodiment of the present invention;

[0039] Fig.15 Schematic diagram of the interaction between NHLLPLR and different amino acid residues of ACE in an embodiment of the present invention;

[0040] Fig.16 Schematic diagram of the conformation of the complex formed after HLPL and ACE are docked in an embodiment of the present invention;

[0041] Fig.17 Schematic diagram of the interaction between HLPL and different amino acid residues of ACE in an embodiment of the present invention;

[0042] Fig.18 Schematic diagram of the conformation of the complex formed after docking of YLSF and DPP4 in an embodiment of the present invention;

[0043] Fig.19 Schematic diagram of the interaction between YLSF and different amino acid residues of DPP4 in the embodiment of the present invention;

[0044] Fig. 20 Schematic diagram of the conformation of the complex formed after docking of SPHWNVN and DPP4 in an embodiment of the present invention;

[0045] Fig.21 Schematic diagram of the interaction between SPHWNVN and different amino acid residues of DPP4 in the embodiment of the present invention;

[0046] Fig. 22 Schematic diagram of the conformation of the complex formed after ESWNPRDPQF and DPP4 docking in an embodiment of the present invention;

[0047] Fig.23 Schematic diagram of the interaction between ESWNPRDPQF and different amino acid residues of DPP4 in the embodiment of the present invention;

[0048] Fig.24 Schematic diagram of the conformation of the complex formed after GNLYSPHWNVN and DPP4 docking in the embodiment of the present invention;

[0049] Fig.25 Schematic diagram of the interaction between GNLYSPHWNVN and different amino acid residues of DPP4 in the embodiment of the present invention;

[0050] Fig.26 Schematic diagram of the conformation of the complex formed after HNLPLLR and DPP4 docking in an embodiment of the present invention;

[0051] Fig. 27 Schematic diagram of the interaction between HNLPLLR and different amino acid residues of DPP4 in the embodiment of the present invention;

[0052] Fig.28 Schematic diagram of the conformation of the complex formed after WNPRDPQ and DPP4 docking in an embodiment of the present invention;

[0053] Fig.29 Schematic diagram of the interaction between WNPRDPQ and different amino acid residues of DPP4 in the embodiment of the present invention;

[0054] Fig.30 Schematic diagram of the conformation of the complex formed after docking of NHLLPLR and DPP4 in an embodiment of the present invention;

[0055] Fig.31 Schematic diagram of the interaction between NHLLPLR and different amino acid residues of DPP4 in the embodiment of the present invention;

[0056] Fig.32 Schematic diagram of the conformation of the complex formed after HLPL and DPP4 docking in an embodiment of the present invention;

[0057] Fig.33 Schematic diagram of the interaction between HLPL and different amino acid residues of DPP4 in an embodiment of the present invention. DETAILED DESCRIPTION

[0058] In this embodiment, a hydrolysis method is first used to prepare a polypeptide derived from bitter almonds as a raw material. The preparation is carried out according to the following method:

[0059] 1. Debitter and peel the almonds, and then perform subcritical degreasing. Select fresh, mildew-free, and insect-free almonds as raw materials, soak them in hot water, and then use a peeling machine to peel them, which can effectively remove the bitterness and skin of the almonds. The solvent used for subcritical degreasing is butane, the material-liquid ratio during subcritical degreasing is 1g / 5mL, the temperature is 50°C, the pressure during subcritical degreasing is 0.52MPa, the degreasing time is 40min / time, and a total of 3 degreasing times. The defatted almond powder obtained in this step needs to be fully dried and stored.

[0060] 2. Removal of impurities from defatted almond powder. Use running water to ultrafilter the defatted almond powder. The water temperature used during ultrafiltration is 4°C. The ultrafiltration membrane used is a 3kDa ultrafiltration membrane. The ultrafiltration time is 24 hours. During ultrafiltration, small molecular impurities (including soluble sugars) are carried away with the water flow, and the defatted almond powder remains in the ultrafiltration membrane. This step is equivalent to removing impurities from the defatted almond powder using running water.

[0061] 3. The defatted almond powder after impurities removal is subjected to alkali dissolution and acid precipitation in turn. The pH of the alkali dissolution is 9.5 (to dissolve the protein in the defatted almond powder initially), and the pH of the acid precipitation is 4 (to promote protein precipitation).

[0062] When alkali-dissolving, first add 20 times the mass of water to the defatted almond powder (the mass of water is calculated by volume, i.e., 20 mL of water is added to 1 g of defatted almond powder), and then add solid sodium hydroxide until the pH reaches 9.5, then stir, centrifuge after 60 minutes, and retain the supernatant. Add 11 mol / L hydrochloric acid to the supernatant until the pH reaches 4, centrifuge again, discard the supernatant, wash the precipitate, and freeze-dry it to obtain almond protein powder.

[0063] 4. Hydrolyze almond protein powder with papain. Add almond protein powder to water to prepare a substrate dispersion with a concentration of 40 mg / mL, adjust the pH to 6.5, add papain to the substrate dispersion, and the enzyme activity of papain required for hydrolysis is 20,000 U / g (i.e., for every 1 g of almond protein powder contained in the substrate dispersion, add 20,000 U of papain), the hydrolysis temperature is 60°C, and the hydrolysis time is 220 minutes. This step obtains almond protein hydrolyzate.

[0064] 5. Use a 3 kDa filter cup to ultrafilter the almond protein hydrolysate in step 4, collect components with a molecular weight less than 3 kDa (i.e., almond peptides with a molecular weight less than 3 kDa) for separation and purification by gel chromatography and C18 reversed phase chromatography.

[0065] During gel chromatography purification, the chromatographic column used is a Sephadex G-15 gel filtration chromatography column (Co l umn XK16 / 70). Before purification begins, the chromatographic column is fully rinsed with a large amount of ultrapure water to balance the column. After reaching column equilibrium, the almond peptide with a molecular weight of less than 3kDa is slowly loaded onto the chromatographic column.

[0066] The GE-AKTA-pure purification system was used, the flow rate was set to 0.5 mL / min, the detection wavelength was set to 280 nm, and ultrapure water was used to elute the chromatographic column to achieve the separation of components in almond peptides with a molecular weight of less than 3 kDa. Due to the characteristics of Sephadex G-15, small molecule peptides will be delayed in elution in the chromatographic column, while larger molecules will pass through faster.

[0067] The elution peak (i.e., absorption peak) on the chromatogram was detected and identified according to the absorbance detection signal intensity at 280 nm, and the detection threshold was set to 50 mAU. Each elution peak was packaged into one tube, and a group of G-15 elution components was obtained.

[0068] The antioxidant capacity of each G-15 elution fraction was evaluated by ABTS+ free radical scavenging test, and it was finally found that the G-15 elution fraction corresponding to the 4# peak had the highest antioxidant capacity. The 4th G-15 elution fraction corresponding to the 4# peak was selected for freeze-drying to obtain the almond peptide purified by gel chromatography. The almond peptide purified by gel chromatography can be stored at -40°C for future use.

[0069] For the almond peptide purified by gel chromatography, C18 reverse chromatography purification was further performed. The model of the C18 chromatographic column used was Ultramate XB-C18 5um 21.2*250mm. Mobile phase A was a trifluoroacetic acid aqueous solution with a trifluoroacetic acid (TFA) volume fraction of 0.02%, and mobile phase B was a trifluoroacetic acid acetonitrile (ACN) solution with a trifluoroacetic acid (TFA) volume fraction of 0.02%.

[0070] During reverse chromatography purification, the almond peptide purified by gel chromatography was dissolved in mobile phase A to a concentration of 50 mg / mL, and injected into a C18 chromatographic column with an injection volume of 5 μL. The column temperature was set to 40°C, the (absorbance) detection wavelength was 220 nm, and the detection threshold was 50 mAU. The elution cycle was 60 min, during which the volume percentage of mobile phase A was linearly reduced from 100% to 0%, while the volume percentage of mobile phase B was linearly increased from 0% to 100%, with a flow rate of 1.0 mL / min. Each elution peak was collected as an elution component, and the solvent was removed by lyophilization to obtain a purified polypeptide sample.

[0071] 6. Perform mass spectrometry sequencing on the purified peptide samples to analyze the peptide sequences. Based on the sequencing results, several peptides with higher content were screened out for biological activity prediction and molecular docking simulation to evaluate their application potential in inhibiting ACE activity and lowering blood sugar.

[0072] For the peptide sequences with high content screened out after sequencing, the BIOPEP-UWM database (https: / / biochemia.uwm.edu.pl / biopep / start_biopep.php) was used to predict their biological functions. Figure 1 The figure shows the biological function prediction results of the peptide sequence YLSF in the BIOPEP-UWM database. In the figure, Activity represents biological function, A represents the score of the corresponding biological function, and the higher the score, the greater the possibility that the peptide has the corresponding function. In the figure, B represents the potential activity of the protein, and the higher the value, the higher the activity of the protein. As can be seen from the figure, the scores of the angiotensin converting enzyme inhibitor (ACE inhibitor) and dipeptidyl peptidase-IV inhibitor (DPP4 inhibitor) of the peptide YLSF are high, indicating that the peptide YLSF is very likely to have the function of inhibiting angiotensin converting enzyme and dipeptidyl peptidase-IV.

[0073] Furthermore, the biological activity of each polypeptide was predicted using the PeptideRanker database (http: / / distildeep.ucd.ie / PeptideRanker / ).

[0074] According to the predicted results of biological activity and biological function, peptides with high biological activity and at least one of the functions of reducing blood pressure and reducing blood sugar were screened out from the sequences of peptides with high content screened out after sequencing, as shown in Table 1, No. 1 to 10. The sequence of each polypeptide, the biological function with a score greater than or equal to 0.5, and the score of the specific biological function are also recorded in Table 1. Among them, angiotensin converting enzyme inhibitors have the effect of lowering blood pressure, and dipeptidyl peptidase-IV inhibitors and serine protease inhibitors have the effect of lowering blood sugar. In addition, antioxidants can inhibit the activity of α-glucosidase and can also play a role in lowering blood sugar.

[0075] Table 1

[0076]

[0077]

[0078] From the biological activity values ​​in Table 1, it can be seen that the biological activities of the two peptides LPSYVN and ESWNPRDPQ are relatively low, so they are discarded. For the other 8 peptides, the molecular docking method was used to test their binding ability with ACE and DPP4 respectively. The following is a further explanation using the test of the binding ability of the peptide with ACE as an example.

[0079] Method for testing the binding ability of peptides to ACE:

[0080] (1) Chemical component collection: Use pymol software (educational version) to construct the peptide 3D structure, and finally save the peptide 3D structure in pdb format.

[0081] (2) Structural acquisition and preprocessing of protein crystals: Download the PDB file of ACE (PDB ID: 1o86) from the PDB database (https: / / www.rcsb.org / ). Then, the protein (ACE) was preprocessed using the discovery studio software to delete water molecules, hydrogenation, and charges, and extract the original ligands in the structure.

[0082] (3) Determination of the active site of the protein: The active site of the protein (ACE) was obtained according to the 13th to 16th lines (“grid box…z inc coordinate”) on the right column of page 5 of the paper “ACE inhibitory peptides from enzyme hydrolysate of fermented black sesame seed: Random forest-based optimization, screening, and molecular docking analysis”.

[0083] (4) Use Autodock-vina for molecular docking: first, put the pre-processed ACE, peptide and vina (software code for docking) into a folder; second, save the prepared peptide 3D structure and pre-processed ACE in pdbqt format, and select ACE as the receptor and the peptide as the ligand; use Notepad to record and save the docking active site, receptor name and ligand name; run vina docking from the command window; use PyMOL to combine the docking results to form a complex.

[0084] The method for testing the binding ability of a polypeptide to DPP4 is the same as the above-mentioned "method for testing the binding ability of a polypeptide to ACE", except that the active site of the protein (DPP4) in step (3) is obtained according to the second paragraph of the right column on page 9 of the paper "Identification of antibiotic peptides derived from in silico hydrolysis of three ancient grains: Amaranth, Quinoa and Chia".

[0085] When performing molecular docking, the software will give binding energy data, and the final binding energy data is shown in Table 2.

[0086] Table 2

[0087] Peptide sequence ACE(kcal / mol) DPP4 (kcal / mol) YLSF -9 -7.4 SPHWNV -9.9 -8.8 ESWNPRDPQF -10.8 -8.2 GNLYSPHWNVN -9.4 -6.6 HNLPLLR -9.3 -7.8 WNPRDP -10.3 -8.3 NHLLPLR -8.1 -8.2 HLPL -8.6 -7.5

[0088] Generally speaking, the lower the binding energy (usually expressed as a larger negative value), the tighter the two substances are bound and the higher the stability of the binding. In addition, the molecular interaction between the peptide and the enzyme (referring to ACE or DPP4) can also reflect the stability of the binding between the two.

[0089] like Figure 2 to Figure 17 It is a schematic diagram of the conformation of the complex formed after the peptide and ACE molecule are docked, and the interaction relationship between the peptide and ACE. The meanings of the English words in the figure are shown in Table 3.

[0090] Table 3

[0091] Conventional Hydrogen Bond Conventional hydrogen bonding interactions van der Waals Van der Waals forces Pi-Sigma Pi-σ interactions Pi-Alkyl Pi-alkyl interactions Carbon Hydrogen Bond Carbon-Hydrogen Bond Unfavorable Donor-Donor Adverse donor-donor interactions Pi-Donor Hydrogen Bond Pi-donor hydrogen bonds Pi-Pi Stacked Pi-Pi Stacking Alkyl Alkyl interactions Pi-Cation Pi-cation interactions Attractive Charge Attraction due to charge difference Unfavorable Acceptor-Acceptor Adverse receptor-receptor interactions Pi-Pi T-shaped Pi-Pi T-shaped interaction Amide-Pi Stacked Amide-Pi stacking Pi-Sulfur Pi-sulfur interactions Salt bridge Salt Bridge Metal-Acceptor Metal-receptor interactions Unfavorable Positive-Positive Unfavorable interactions between positive charges

[0092] like Figure 2 This is the conformation of the complex formed after docking of YLSF and ACE. In the right part of the figure, blue represents nitrogen, red represents oxygen, yellow represents carbon in the peptide YLSF, and green represents carbon in ACE; Figure 3 The interaction between peptide YLSF and different amino acid residues of ACE is shown. Figure 3 It can be seen that after docking, multiple hydrogen bonds were formed between YLSF and ACE, and there were van der Waals forces, Pi-σ interactions, and Pi-alkyl interactions, indicating that YLSF can stably bind to ACE.

[0093] like Figure 4 The conformation of the complex formed after SPHWNVN and ACE docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Figure 5 The interaction between the peptide SPHWNVN and different amino acid residues of ACE is shown. Figure 5It can be seen that after docking, SPHWNVN formed multiple hydrogen bonds and carbon-hydrogen bonds with ACE, and there were various types of interactions, with fewer unfavorable donor-donor interactions, indicating that SPHWNVN can stably bind to ACE.

[0094] like Figure 6 The conformation of the complex formed after ESWNPRDPQF and ACE docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Figure 7 The interaction between ESWNPRDPQF and different amino acid residues of ACE is shown. Figure 7 It can be seen that after docking, ESWNPRDPQF formed multiple hydrogen bonds and one carbon-hydrogen bond with ACE, and there were multiple types of interactions, with fewer unfavorable receptor-receptor interactions, indicating that ESWNPRDPQF can stably bind to ACE.

[0095] like Figure 8 The conformation of the complex formed after GNLYSPHWNVN and ACE docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Fig. 9 The interaction between GNLYSPHWNVN and different amino acid residues of ACE is shown. Fig. 9 It can be seen that after docking, GNLYSPHWNVN formed multiple hydrogen bonds and carbon-hydrogen bonds with ACE, and there were various types of interactions, with fewer unfavorable donor-donor interactions, indicating that GNLYSPHWNVN can stably bind to ACE.

[0096] like Fig.10 The conformation of the complex formed after HNLPLLR and ACE docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 .Depend on Fig.11 It can be seen that after HNLPLLR docked with ACE, multiple hydrogen bonds and carbon-hydrogen bonds were formed, and there were various types of interactions, indicating that HNLPLLR can stably bind to ACE.

[0097] like Fig.12 The conformation of the complex formed after WNPRDPQ and ACE docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Fig.13 The interaction between WNPRDPQ and different amino acid residues of ACE is shown. Fig.13 It can be seen that after docking, multiple hydrogen bonds and one carbon-hydrogen bond were formed between WNPRDPQ and ACE, and there were multiple types of interactions, indicating that WNPRDPQ can stably bind to ACE.

[0098] like Fig.14The conformation of the complex formed after NHLLPLR and ACE docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Fig.15 The interaction between NHLLPLR and different amino acid residues of ACE is shown. Fig.15 It can be seen that after docking, NHLLPLR formed multiple hydrogen bonds and one carbon-hydrogen bond with ACE, and there were multiple types of interactions, indicating that NHLLPLR can stably bind to ACE.

[0099] like Fig.16 The conformation of the complex formed after HLPL and ACE docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Fig.17 The interaction between HLPL and different amino acid residues of ACE is shown. Fig.17 It can be seen that multiple hydrogen bonds were formed after HLPL docked with ACE, and there were various types of interactions, indicating that HLPL can stably bind to ACE.

[0100] like Figures 18 to 31 It is a schematic diagram of the conformation of the complex formed after the peptide and DPP4 molecule are docked and the interaction relationship between the peptide and DPP4. The meanings of the English characters in the figure are the same as those in Table 3.

[0101] like Fig.18 The conformation of the complex formed after docking of YLSF and DPP4. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Fig.19 The interaction between YLSF and different amino acid residues of DPP4 is shown. Fig.19 It can be seen that there are various types of interactions such as hydrogen bonds between YLSF and DPP4, indicating that YLSF can bind to DPP4.

[0102] like Fig. 20 The conformation of the complex formed after SPHWNVN and DPP4 docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Fig.21 The interaction between the peptide SPHWNVN and different amino acid residues of DPP4 is shown. Fig.21 It can be seen that after docking, SPHWNVN formed multiple hydrogen bonds and one carbon-hydrogen bond with DPP4, and there were multiple types of interactions, with fewer unfavorable donor-donor interactions, indicating that SPHWNVN can stably bind to DPP4.

[0103] like Fig. 22 The conformation of the complex formed after ESWNPRDPQF and DPP4 docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Fig.23The interaction between ESWNPRDPQF and different amino acid residues of DPP4 is shown. Fig.23 It can be seen that after docking, ESWNPRDPQF formed multiple hydrogen bonds with DPP4, and there were multiple types of interactions, with fewer unfavorable receptor-receptor interactions, indicating that ESWNPRDPQF can stably bind to DPP4.

[0104] like Fig.24 The conformation of the complex formed after GNLYSPHWNVN and DPP4 docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Fig.25 The interaction between GNLYSPHWNVN and different amino acid residues of DPP4 is shown. Fig.25 It can be seen that after docking, GNLYSPHWNVN formed multiple hydrogen bonds and carbon-hydrogen bonds with DPP4, and there were various types of interactions, with fewer unfavorable donor-donor interactions, indicating that GNLYSPHWNVN can stably bind to DPP4.

[0105] like Fig.26 The conformation of the complex formed after HNLPLLR and DPP4 docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Fig. 27 The interaction between HNLPLLR and different amino acid residues of DPP4 is shown. Fig. 27 It can be seen that after docking, HNLPLLR formed multiple hydrogen bonds and one carbon-hydrogen bond with DPP4, and there were multiple types of interactions, indicating that HNLPLLR can stably bind to DPP4.

[0106] like Fig.28 The conformation of the complex formed after WNPRDPQ and DPP4 docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Fig.29 The interaction between WNPRDPQ and different amino acid residues of DPP4 is shown. Fig.29 It can be seen that after docking, WNPRDPQ formed multiple hydrogen bonds with DPP4, and there were multiple types of interactions, indicating that WNPRDPQ can stably bind to DPP4.

[0107] like Fig.30 The conformation of the complex formed after NHLLPLR and DPP4 docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Fig.31 The interaction between NHLLPLR and different amino acid residues of DPP4 is shown. Fig.31It can be seen that after docking, NHLLPLR formed multiple hydrogen bonds and carbon-hydrogen bonds with DPP4, and there were various types of interactions, indicating that NHLLPLR can stably bind to DPP4.

[0108] like Fig.32 The conformation of the complex formed after HLPL and DPP4 docking. The elements represented by the colors on the right side of the figure are the same. Figure 2 . Fig.33 The interaction between HLPL and different amino acid residues of DPP4 is shown. Fig.33 It can be seen that after docking, HLPL and DPP4 formed multiple hydrogen bonds and there were various types of interactions, indicating that HLPL can stably bind to DPP4.

[0109] Taking into account the size of the binding energy, the biological activity of the polypeptide itself, and the predicted biological function of the polypeptide, the inventors further conducted confirmatory tests on the inhibitory properties of the two polypeptides YSLF and WNPRDPQ against ACE and DPP4, and conducted confirmatory tests on the inhibitory properties of HLPL against DPP4.

[0110] The specific method for detecting the inhibitory performance of YSLF and WNPRDPQ on ACE is as follows:

[0111] Equipment and materials: 0.1 mol / L boric acid buffer (containing 0.3 mol / L NaCl, pH 8.3), Angiotensin-I-Converting Enzyme (ACE), substrate hippury l-Lhistidy lL-leucine (HHL, hippurylhistidylleucine), HCl, ethyl acetate, teprotide, water bath, drying oven, and spectrophotometer.

[0112] Testing steps:

[0113] (1) Use borate buffer (BBS) to dilute ACE and HHL to 20 mU / mL and 10 mmol / L, respectively.

[0114] (2) YSLF and WNPRDPQ as samples and teprotide as a positive control were diluted to 1000 μg / mL using BBS, and then diluted in a 1 / 4 gradient until 5 concentration gradients were obtained.

[0115] (3) For each sample with gradient concentration, 50 μL sample + 150 μL substrate + 50 μL ACE were mixed; for each sample with gradient concentration, 50 μL sample + 150 μL substrate + 50 μL BBS were mixed as sample control; negative control was 50 μL BBS + 150 μL substrate + 50 μL ACE; blank control was 50 μL BBS + 150 μL substrate + 50 μL BBS; positive control was 50 μL teprotide (concentration was the same as sample concentration) + 150 μL substrate + 50 μL ACE.

[0116] (4) Incubate the various mixed solutions obtained in step (3) at 37°C for 120 minutes.

[0117] (5) Add 250 μL of 1 mmol / L HCl to the mixture to terminate the reaction.

[0118] (6) Add 1 mL of ethyl acetate to extract the reaction product, hippuric acid, and take the supernatant. Repeat this process 3 times.

[0119] (7) Ethyl acetate was evaporated at 60°C.

[0120] (8) After evaporating the ethyl acetate, the residue (hippuric acid) was dissolved by adding 2 mL of ddH2O (heating required), and then measured by spectrophotometer at 228 nm. The specific concentration of hippuric acid was calculated by the standard curve method.

[0121] The inhibition rate was calculated according to the following formula:

[0122] ACE-inh ibitory activity(%)={1-[(AB)-(A C -B)] / (CB)}×100%

[0123] A: sample measurement value; B: blank control; C: negative control; A C : Sample control

[0124] The measurement data of the sample control are shown in Table 4.

[0125] Table 4

[0126]

[0127] The measured values ​​of the samples and the calculated data of the inhibition rate are shown in Table 5. The calculation method of the value of "mean-blank-sample control" in Table 5 is the same as [(AB)-(A C -B)], IC50 in the table represents the concentration of the inhibitor (teprotide, YLSF or WNPRDPQ) when the reaction of ACE-catalyzed substrate decomposition is inhibited by half.

[0128] Table 5

[0129]

[0130]

[0131] As can be seen from the results in the table, with the increase of concentration, these three substances all show an increasing trend in the inhibition rate of ACE. However, when the concentration of teprotide is low (less than or equal to 62.5 μg / mL), the inhibition rate of teprotide varies little with the change of concentration, indicating that when the concentration of teprotide is low, its effect is less affected by the change of concentration. However, for YLSF and WNPRDPQ, even at low concentrations, the magnitude of its inhibitory effect on ACE shows a nearly linear correlation with its own concentration. This feature enables YLSF and WNPRDPQ to adapt to some special usage scenarios. For example, in scientific research experiments, when the activity of ACE needs to be lowered by about 35% to 50%, it is difficult to use teprotide, while YLSF and WNPRDPQ in this embodiment can have an effect.

[0132] When determining the inhibition rate of YSLF and WNPRDPQ on DPP4, the Dipeptidyl peptidase IV (DPP4) inhibitor screening assay kit (Abcam product, catalog number ab133081) was used to detect the inhibitory effects of the two on DPP4. The specific test method is shown in the kit manual.

[0133] For the two peptides YSLF and WNPRDPQ, the diluent in the kit was used to adjust the stock solution to 1 mM, and the 1 / 4 gradient dilution was 250, 62.5, 15.625, 3.906 μM. The final concentration of the peptide in the reaction system was 100, 25, 6.25, 1.5625, 0.3906 μM. In the positive control, the final concentration of the standard inhibitor (sitagliptin) was 100, 25, 6.25, 1.5625, 0.3906 μM. The negative control contained DPP4 and the reaction substrate, and the blank control contained only the reaction substrate without DPP4.

[0134] The test results are shown in Table 6, where IC50 represents the concentration of the inhibitor (sitagliptin, YLSF or WNPRDPQ) when the reaction of DPP4-catalyzed substrate decomposition is inhibited by half. As shown in Table 6, both YSLF and WNPRDPQ can inhibit the activity of DPP4 to a certain extent.

[0135] Table 6

[0136]

[0137]

[0138] When measuring the inhibition rate of HLPL on DPP4, the Dipeptidy l peptidase IV (DPP4) inhibitor screening assay kit (Abcam product, catalog number ab133081) was used to detect the inhibitory effect of HLPL on DPP4.

[0139] Use the diluent in the kit to dilute HLPL to 200mM, and then dilute it in a 1 / 5 gradient to 40mM, 8mM, 1.6mM, and 0.32mM. The final concentrations of HLPL in the reaction system are 20mM, 4mM, 0.8mM, 0.16mM, and 0.032mM, respectively; the final concentrations of the standard inhibitor (sitagliptin) in the positive control are 100μM, 25μM, 6.25μM, 1.563μM, and 0.391μM, respectively.

[0140] The specific method of the test is shown in the kit instructions. The test results are shown in Table 7, where IC50 represents the concentration of the inhibitor (sitagliptin or HLPL) when the reaction of DPP4-catalyzed substrate decomposition is inhibited by half.

[0141] Table 7

[0142]

[0143] From the above results, it can be seen that HLPL has obvious DPP4 inhibitory activity, that is, HLPL can inhibit the function of DPP4.

[0144] The above YLSF, WNPRDPQ and HLPL are expected to be used to develop polypeptide drugs with dual effects of lowering blood pressure and lowering blood sugar, and the preparation methods of the above YLSF, WNPRDPQ and HLPL are simple, and the raw materials are bitter almonds, which are low in cost. That is, the use of bitter almonds to prepare polypeptide drugs with dual effects of lowering blood pressure and lowering blood sugar has broad market and application prospects.

[0145] Furthermore, the inventors have determined the conditions for preparing and isolating the above 8 peptides with antihypertensive and hypoglycemic effects verified by molecular docking experiments from almond peptides purified by gel chromatography. The preparative separation uses a Biotage rapid preparative liquid chromatograph, the chromatographic column is Teledyne Isco, 150 gram HP C18, the column volume (CV): 130 mL, the column temperature is 40 ° C, the mobile phase A is water, the mobile phase B is acetonitrile; the flow rate is 60 mL / min, the (absorbance) detection wavelength is 220 nm, and the detection threshold is 50 mAU.

[0146] The elution cycle is 60 minutes. During the elution cycle, the volume percentage of ultrapure water is linearly reduced from 100% to 0%, while the volume percentage of acetonitrile is linearly increased from 0% to 100%; each elution peak (absorption peak) is collected as a component. Mass spectrometry sequencing is performed on each component, and the peptide chain with the correct sequencing is selected.

[0147] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A polypeptide having the effects of lowering blood pressure and blood sugar, characterized in that: The amino acid sequence of the polypeptide is shown in SEQ ID NO.1, or SEQ ID NO.3, or SEQ ID NO.4, or SEQ ID NO.5, or SEQ ID NO.6, or SEQ ID NO.8, or SEQ ID NO.9, or SEQ ID NO.

10.

2. The polypeptide according to claim 1, characterized in that The polypeptide with the amino acid sequence shown in SEQ ID NO.1 and the polypeptide with the amino acid sequence shown in SEQ ID NO.8 are both polypeptides having the effect of lowering blood pressure, and the polypeptide with the amino acid sequence shown in SEQ ID NO.10 is a polypeptide having the effect of lowering blood sugar.

3. The use of a polypeptide, characterized in that: The application is to use any one of the polypeptides described in claim 1 or 2 for manufacturing a drug having the function of lowering blood pressure and / or blood sugar.

4. The use according to claim 3, characterized in that: The application is to use the polypeptide with amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.8 to manufacture a drug with blood pressure lowering function, and to use the polypeptide with amino acid sequence as shown in SEQ ID NO.10 to manufacture a drug with blood sugar lowering function.

5. A method for preparing a polypeptide, characterized in that: The following steps are involved: S1, debittering, peeling and crushing almonds, and then subcritical defatting to obtain defatted almond powder; S2. Using running water to remove impurities from defatted almond flour; S3, dissolving the defatted almond powder after impurities removal with alkali, then removing insoluble matter, subjecting the solution to acid precipitation, washing and drying the precipitate obtained by the acid precipitation, and obtaining almond protein powder; S4, hydrolyzing the almond protein powder using a protease to obtain an almond protein hydrolyzate; S5. Purify and freeze-dry the almond protein hydrolysate to obtain the polypeptide as claimed in claim 1 or 2.

6. The preparation method according to claim 5, characterized in that: In step S1, the solvent used in subcritical degreasing is butane; in step S2, when removing impurities, running water is used to ultrafilter the defatted almond powder; the ultrafiltration membrane used in ultrafiltration is a 3kDA ultrafiltration membrane, the water temperature during ultrafiltration is 4-10°C, and the ultrafiltration time is 22-26 hours.

7. The preparation method according to claim 5, characterized in that: In step S3, during alkali dissolution, the mass / volume ratio of defatted almond powder to the alkali dissolution reaction system is 1g:(15-20)mL, the pH of the alkali dissolution reaction system is 8.8-9.5, and the alkali dissolution time is 1-3 hours; the acid used during acid precipitation is hydrochloric acid, the concentration of hydrochloric acid is 8-12 mol / L, and the pH of the acid precipitation reaction system is 3.3-4.

7.

8. The preparation method according to claim 5, characterized in that: In step S4, papain is used to hydrolyze almond protein powder; in the hydrolysis reaction system, the concentration of almond protein powder is 30-50 mg / mL, and the mass ratio of papain to almond protein powder is 18000-20000 U / g; the pH of the hydrolysis reaction system is 6.5-7.2, the temperature of the hydrolysis reaction is 55-65° C., and the time of the hydrolysis reaction is 3-4 hours.

9. The preparation method according to claim 8, characterized in that: In the hydrolysis reaction system, the concentration of almond protein powder is 40 mg / mL, the mass ratio of papain to almond protein powder is 20000 U / g; the pH of the hydrolysis reaction system is 6.5, the hydrolysis temperature is 60° C., and the hydrolysis time is 220 minutes.

10. The preparation method according to claim 5, characterized in that: In step S5, when purifying the almond protein hydrolysate, ultrafiltration is used to remove the components with a molecular weight less than 3 kDa in the almond protein hydrolysate, and then the remaining components are purified by gel chromatography and reverse phase chromatography in sequence.

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