Cicada pupa polypeptide with ACE inhibitory activity as well as preparation method and application of cicada pupa polypeptide

Abstract

The invention discloses a cicada pupa polypeptide with ACE inhibitory activity and a preparation method and application thereof, and belongs to the technical field of functional food. According to the method, cicada pupa protein is used as a raw material, after enzymolysis, ultrafiltration, separation and screening, sequencing and molecular docking verification, four cicada pupa protein polypeptides with ACE inhibitory activity are obtained, and the amino acid sequences of the four cicada pupa protein polypeptides are Ser-Tyr-Arg-Phe, Gly-Pro-Lys-Leu-Phe, Ser-Pro-Arg-Pro-Trp and Phe-Arg-Gly-Phe respectively. The polypeptide obtained by the invention has very strong ACE inhibitory activity, small influence on cell proliferation, low cytotoxicity and high safety, can be used for development and preparation of functional food or drugs for reducing blood pressure, and has good application prospects.

Description

Technical Field

[0001] The invention relates to a cicada pupa polypeptide with ACE inhibitory activity and a preparation method and application thereof, belonging to the technical field of functional foods. Background Art

[0002] Hypertension is a chronic disease with high morbidity, high disability rate and high mortality rate, which has seriously threatened human life safety. Various external and internal factors can affect the changes in human blood pressure, but the balance between the pressor system - renin-angiotensin system (RAS) and the antihypertensive system - kallikrein-kinin system (KKS) is the most important regulatory system. ACE in the RAS system is an important factor causing high blood pressure. Therefore, starting from inhibiting the activity of ACE, developing ACE inhibitor drugs is an effective way to treat hypertension. At present, there are many angiotensin converting enzyme inhibitors (ACEI) (such as "captopril", "lisinopril", etc.) that have been widely used in the clinical treatment of hypertension. This type of drug has a significant antihypertensive effect in clinical practice, but it also produces some side effects.

[0003] At present, food-borne ACE inhibitory peptides are usually obtained from proteins by enzymatic hydrolysis, and peptides with ACE inhibitory activity are obtained after separation, purification, and mass spectrometry identification. Previous studies have found that the inhibition types of ACE inhibitory peptides include competitive inhibitors, non-competitive inhibitors, and mixed inhibitors, but peptides in competitive inhibition mode usually have stronger ACE inhibitory activity. When ACE inhibitory peptides were applied to human umbilical vein epithelial cells (HUVEC), it was found that the peptides could increase the secretion of cellular NO and reduce the production of ET-1. It is speculated that ACE inhibitory peptides can achieve the purpose of lowering blood pressure in this way.

[0004] Cicada pupae are a typical high-protein insect, with a protein content of up to 72%. At present, silkworm pupa protein peptides have been proven to have significant biological activities in anti-oxidation, uric acid reduction and anti-inflammation. As a nutritious insect resource, cicada pupae also have high development and utilization potential. However, research on the functional activity of cicada pupa protein is still insufficient and needs further exploration.

[0005] Therefore, functional peptides with ACE inhibitory activity derived from animal and plant proteins and natural organisms have attracted widespread attention due to their safety and lack of side effects. Exploring functional peptides with ACE inhibitory activity is of great significance for the development of safer and more effective drugs or functional foods. Summary of the invention

[0006] In order to solve the defects of the prior art, the present invention provides a cicada pupa polypeptide with ACE inhibitory activity and its preparation method and application. The polypeptide obtained by using high-quality insect protein cicada pupa protein as raw material has strong ACE inhibitory activity, little effect on cell proliferation, low cytotoxicity, high safety, and can be used for the development of blood pressure lowering functional foods or medicines, with broad application prospects.

[0007] The invention provides a cicada pupa protein peptide with ACE inhibitory activity. The amino acid sequence of the protein peptide is Ser-Tyr-Arg-Phe, Gly-Pro-Lys-Leu-Phe, Ser-Pro-Arg-Pro-Trp or Phe-Arg-Gly-Phe, as shown in SEQ ID NOs.1-4.

[0008] SEQ ID NO.1: SYRF;

[0009] SEQ ID NO.2: GPKLF;

[0010] SEQ ID NO.3: SPRPW;

[0011] SEQ ID NO.4: FRGF.

[0012] In one embodiment of the present invention, the cicada pupa protein peptide has a strong inhibitory effect on ACE activity; wherein, the half inhibitory concentration (IC50) of Ser-Tyr-Arg-Phe is 0.026 mg / mL; the half inhibitory concentration (IC50) of Gly-Pro-Lys-Leu-Phe is 0.118 mg / mL; the half inhibitory concentration (IC50) of Ser-Pro-Arg-Pro-Trp is 0.34 mg / mL; the half inhibitory concentration (IC50) of Phe-Arg-Gly-Phe is 1.716 mg / mL.

[0013] In one embodiment of the present invention, the cicada pupa protein peptide can be used as food, medicine or health product; it is used to inhibit ACE and lower blood pressure, and has good application prospects as a health product and drug lead compound for hypertension, heart disease or cardiovascular disease.

[0014] In one embodiment of the present invention, the cicada pupa protein peptide is prepared using cicada pupae as raw material.

[0015] In one embodiment of the present invention, the method for preparing cicada pupa protein peptide comprises the steps of:

[0016] Using dried defatted cicada pupae as raw materials, four polypeptides with ACE inhibitory activity were obtained after enzymatic hydrolysis, ultrafiltration, separation screening, sequencing and molecular docking verification. The amino acid sequences were Ser-Tyr-Arg-Phe, Gly-Pro-Lys-Leu-Phe, Ser-Pro-Arg-Pro-Trp and Phe-Arg-Gly-Phe, respectively.

[0017] (1) defatting cicada pupa powder, adding NaOH to extract, centrifuging and discarding the precipitate to obtain cicada pupa protein alkaline extract; adjusting pH, standing; centrifuging to obtain cicada pupa protein precipitate, and freeze-drying; adding enzyme for enzymolysis, inactivating the enzyme, centrifuging and discarding the precipitate to obtain cicada pupa protein hydrolysate;

[0018] (2) diluting the cicada pupa protein hydrolysate obtained in step (1) and performing ultrafiltration using a 1 kDa ultrafiltration membrane to obtain a component ≤ 1 kDa, and lyophilizing the component to obtain a lyophilized powder;

[0019] (3) preparing a solution from the lyophilized powder obtained in step (2), and separating the cicada pupa ACE inhibitory peptide by high performance liquid chromatography; collecting multiple components according to the peak elution situation and time, and screening them for ACE inhibitory activity to obtain the component with the strongest ACE inhibitory activity;

[0020] (4) Identifying and screening the components obtained in step (3) to obtain peptides with ACE inhibitory activity.

[0021] In one embodiment of the present invention, the concentration of NaOH in step (1) is 0.05-0.075 mol / L; the temperature during the extraction with NaOH is 50-60°C, and the extraction time is 3.0-4.0 h; the solid-liquid ratio of the defatted cicada pupa powder to NaOH is 1:(20-25) (w / v).

[0022] In one embodiment of the present invention, in step (1), the pH is adjusted to 4.2; and the centrifugation condition is 4000 rpm at 4°C for 20 min.

[0023] In one embodiment of the present invention, the conditions for enzymatic hydrolysis in step (1) are a temperature of 55-60°C, a pH of 9.5-10.0, and an enzymatic hydrolysis time of 3-4 hours; the enzyme used for the enzymatic hydrolysis is alkaline protease, and the enzyme addition amount is 800-1000 U / g; and the enzyme inactivation conditions are 100°C for 10 minutes.

[0024] In one embodiment of the present invention, step (4) is specifically as follows: the components obtained in step (3) are subjected to polypeptide amino acid sequence determination by nano liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS) to obtain enzymatic peptide spectrum information; peptides with a confidence score greater than 80% in the peptide spectrum are scored by Peptide Ranker, and the BIOPEP database is used to predict the ACE inhibitory activity of the peptides; the toxicity, solubility, sensitization and absorption characteristics of the peptides in the human intestine are predicted by ToxinPred, INNOVAGEN, Allergen FP and ADMET Lab 3.0 tools, thereby identifying potential cicada pupa polypeptides with ACE inhibitory activity.

[0025] The present invention also provides an expression vector or a recombinant microorganism, wherein the expression vector or the recombinant microorganism comprises at least one of the above-mentioned cicada pupa protein peptides.

[0026] In one embodiment of the present invention, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector.

[0027] In one embodiment of the present invention, the recombinant microorganism is a bacterium or a fungus.

[0028] The present invention also provides a pharmaceutical composition, which contains a therapeutically effective amount of active ingredients and pharmaceutically acceptable pharmaceutical excipients; the active ingredients include any one or more of the above-mentioned cicada pupa protein peptides: Ser-Tyr-Arg-Phe, Gly-Pro-Lys-Leu-Phe, Ser-Pro-Arg-Pro-Trp or Phe-Arg-Gly-Phe;

[0029] The above-mentioned cicada pupa protein peptide can significantly inhibit the activity of ACE.

[0030] In one embodiment of the present invention, the pharmaceutical excipient refers to a conventional drug carrier in the pharmaceutical field.

[0031] In one embodiment of the present invention, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch, sucrose, etc.; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch, etc.; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters, etc.; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol, etc.; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate, etc.; in addition, other auxiliary agents such as flavoring agents, sweeteners, etc. can also be added to the composition.

[0032] The present invention also provides a food, medicine, health product or nutritional product, which contains an effective dose of at least one of the above-mentioned cicada pupa protein peptides.

[0033] In one embodiment of the present invention, the amino acid sequence of the cicada pupa protein peptide is Ser-Tyr-Arg-Phe, Gly-Pro-Lys-Leu-Phe, Ser-Pro-Arg-Pro-Trp or Phe-Arg-Gly-Phe.

[0034] In one embodiment of the present invention, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0035] In one embodiment of the present invention, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary materials such as flavoring agents and sweeteners can be added to the composition.

[0036] In one embodiment of the present invention, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form.

[0037] In one embodiment of the present invention, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, and oral suspensions.

[0038] In one embodiment of the present invention, the injection dosage form includes but is not limited to injection solution and injection powder.

[0039] In one embodiment of the present invention, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

[0040] In one embodiment of the present invention, the food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods.

[0041] In one embodiment of the present invention, the health care product further contains acceptable excipients.

[0042] The present invention also provides the use of at least one or more of the above-mentioned cicada pupa protein peptides in the preparation of food, medicine, health care products or nutritional products; it is used to inhibit ACE activity, lower blood pressure, prevent or treat hypertension, heart disease or cardiovascular disease.

[0043] In one embodiment of the present invention, the amino acid sequence of the cicada pupa protein peptide is Ser-Tyr-Arg-Phe, Gly-Pro-Lys-Leu-Phe, Ser-Pro-Arg-Pro-Trp or Phe-Arg-Gly-Phe.

[0044] In one embodiment of the present invention, the drug further contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field.

[0045] In one embodiment of the present invention, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary materials such as flavoring agents and sweeteners can be added to the composition.

[0046] In one embodiment of the present invention, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form.

[0047] In one embodiment of the present invention, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, and oral suspensions.

[0048] In one embodiment of the present invention, the injection dosage form includes but is not limited to injection solution and injection powder.

[0049] In one embodiment of the present invention, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

[0050] In one embodiment of the present invention, the food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods.

[0051] In one embodiment of the present invention, the health care product further contains acceptable excipients.

[0052] The present invention also provides a method for preparing the above-mentioned food, medicine, health product or nutritional product, which comprises mixing at least one of the above-mentioned cicada pupa protein peptides with at least one acceptable auxiliary material.

[0053] In one embodiment of the present invention, the amino acid sequence of the cicada pupa protein peptide is Ser-Tyr-Arg-Phe, Gly-Pro-Lys-Leu-Phe, Ser-Pro-Arg-Pro-Trp or Phe-Arg-Gly-Phe.

[0054] Beneficial effects:

[0055] (1) The present invention obtains a cicada pupa polypeptide with ACE inhibitory activity, the specific sequence of which is Ser-Tyr-Arg-Phe, Gly-Pro-Lys-Leu-Phe, Ser-Pro-Arg-Pro-Trp and Phe-Arg-Gly-Phe. It has a novel structure and is easy to prepare. It can be obtained by enzymatic hydrolysis of cicada pupa proteins or artificially synthesized.

[0056] (2) Experiments show that the four polypeptides obtained by the present invention have strong ACE inhibitory activity; among them, the half inhibitory concentration (IC50) of Ser-Tyr-Arg-Phe is 0.026 mg / mL; the half inhibitory concentration (IC50) of Gly-Pro-Lys-Leu-Phe is 0.118 mg / mL; the half inhibitory concentration (IC50) of Ser-Pro-Arg-Pro-Trp is 0.34 mg / mL; and the half inhibitory concentration (IC50) of Phe-Arg-Gly-Phe is 1.716 mg / mL, which have broad application prospects in the field of functional foods.

[0057] (3) The four polypeptides Ser-Tyr-Arg-Phe, Gly-Pro-Lys-Leu-Phe, Ser-Pro-Arg-Pro-Trp and Phe-Arg-Gly-Phe obtained by the present invention have little effect on cell proliferation, low cytotoxicity and high safety.

[0058] (4) The ACE inhibitory peptide of the present invention is derived from cicada pupa protein, which is a high-protein, low-fat, easily digestible high-quality protein source with biological activities such as anti-oxidation, blood pressure reduction, and immunomodulation, and is rich in trace elements. It has a short production cycle, abundant resources, low allergy, high efficiency and sustainability, and is an ideal alternative to traditional proteins with broad market application potential. The present invention provides a scientific basis for the application of cicada pupa protein in the food nutrition and health industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The figures are high performance liquid chromatograms and the results of ACE inhibitory activity test of each component; (A) Preparative high performance liquid chromatogram of component C3; (B) ACE inhibitory activity of each component;

[0060] Figure 2 Mass spectra of ACE inhibitory peptides; (A) FRGF; (B) SPRPW; (C) GPKLF; (D) SYRF;

[0061] Figure 3 The global image and intersection details of molecular docking; (A) SYRF; (B) GPKLF, (C) SPRPW; (D) FRGF;

[0062] Figure 4 Is the Linewaver-Burk diagram; (A) FRGF; (B) SPRPW; (C) GPKLF; (D) SYRF;

[0063] Figure 5 ITC heat flow curves of active peptide titration of ACE; (A) FRGF; (B) SPRPW; (C) GPKLF; (D) SYRF;

[0064] Figure 6 The results of NO and ET-1 secretion of HUVEC cells under different treatment conditions; (A) NO; (B) ET-1;

[0065] Figure 7 The figure shows the survival rate of HUVEC cells under different treatment conditions. DETAILED DESCRIPTION

[0066] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that the examples are intended to better explain the present invention and are not intended to limit the present invention.

[0067] In the following examples, unless otherwise specified, all solutions mentioned use water as the solvent.

[0068] 1. Determination of ACE inhibitory activity

[0069] Preparation of 50 mU / mL ACE solution: Dissolve ACE standard (purchased from Sigma-Aldrich, USA, product number A6778) in 0.1 mol / L boric acid-borax buffer (pH 8.3, containing 0.3 mol / L NaCl) to prepare 50 mU / mL ACE solution.

[0070] Preparation of 7.6mmol / L HHL (maleuconamide) solution: HHL standard (purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S23032) was dissolved in 0.1mol / L boric acid-borax buffer (pH 8.3, containing 0.3mol / LNaCl) to prepare 7.6mmol / L HHL solution.

[0071] Take 20 μL of 1 mg / mL sample solution in a 1.5 mL centrifuge tube, add 20 μL of 50 mU / mL ACE solution, mix well and keep warm in a 37°C water bath for 10 min, then add 40 μL of 7.6 mmol / L HHL solution, mix well and react in a 37°C water bath for 40 min, and finally add 50 μL of 1 mol / L hydrochloric acid to terminate the reaction.

[0072] 0.5 mg / mL hippuric acid was used as a standard sample to determine the retention time of the hippuric acid peak, and 0.1 mol / L boric acid-borax buffer (pH 8.3, containing 0.3 mol / L NaCl) was used instead of the sample solution for reaction as a blank control.

[0073] The content of hippuric acid (HA) in the reaction system was detected by high performance liquid chromatography, using a Waters XBridgeC18 (250 mm×4.6 mm, 5 μm) chromatographic column, a detection wavelength of 238 nm, an injection volume of 5 μL, a flow rate of 0.6 mL / min; mobile phase A was 10% acetonitrile (0.1% TFA), and mobile phase B was pure acetonitrile; column temperature: 40°C.

[0074]

[0075] 2. Peptide content determination method

[0076] Weigh 500 mg of cicada pupa protein hydrolysate in a 25 mL volumetric flask, dilute to 25 mL with 15% trichloroacetic acid solution, centrifuge and take the supernatant, and determine the protein content of the supernatant by the Kjeldahl nitrogen method in accordance with GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Food" to obtain the acid-soluble protein content X1. Weigh 400 mg of cicada pupa protein hydrolysate, dilute to 10 mL with 5% trichloroacetic acid solution, and determine the content of free amino acids in cicada pupa protein hydrolysate X2 by referring to the determination method of free amino acids in GB 22492-2008 "Soybean Peptide Powder".

[0077] Peptide content (g / 100g) = X1-X2;

[0078] Where X1 represents the content of acid-soluble protein, in g / 100g;

[0079] Wherein X2 represents the content of free amino acids, and the unit is g / 100g.

[0080] The endothelial cell basal culture medium involved in the following examples was purchased from Shanghai Anwei Biotechnology Co., Ltd. with the product number of PriMed-AW-002.

[0081] Example 1 Preparation of cicada pupa polypeptide with ACE inhibitory activity

[0082] 1. Enzymatic hydrolysis of cicada pupa

[0083] After defatting the cicada pupa powder, it was extracted for 3.0 h under the conditions of reaction temperature of 60°C, NaOH concentration of 0.075 mol / L, and solid-liquid ratio of 1:25 (w / v), and centrifuged at 4000 rpm for 20 min at 4°C to obtain cicada pupa protein alkaline extract, and the pH of the alkaline extract was adjusted to 4.2 (isoelectric point of cicada pupa protein) with 1 mol / L HCl, and allowed to stand at 4°C overnight, and centrifuged at 4000 rpm for 20 min to obtain cicada pupa protein precipitate, which was freeze-dried to obtain cicada pupa protein (CCP), with a CCP protein content of 81.8 g / 100 g.

[0084] CCP was hydrolyzed under the conditions of hydrolysis temperature of 60°C, pH 9.5, hydrolysis time of 3h, substrate concentration (the ratio of protein mass in the sample to the total mass of the mixture) of 4%, and alkaline protease 6L (purchased from DuPont, USA) added at an enzyme amount of 1000U / g. After the reaction, the enzyme was inactivated at 100°C for 10min, and the precipitate was discarded after centrifugation at 8000rpm for 20min at 4°C to obtain cicada pupa protein hydrolysate (CPPH). According to the above "2. Peptide content determination method", the peptide content was measured to be 82.8%.

[0085] 2. Isolation and purification of cicada pupa polypeptide

[0086] The cicada pupa protein hydrolysate (CPPH) obtained in step 1 was diluted and ultrafiltered using 3kDa and 1kDa ultrafiltration membranes, and the filtrates were collected to obtain three components: C1 (M≥3kDa), C2 (3kDa>M>1kDa), and C3 (M≤1kDa) (M is the molecular weight of the component).

[0087] The C1, C2 and C3 components obtained by ultrafiltration were subjected to rotary evaporation and vacuum freeze drying to prepare freeze-dried powders, which were dissolved in 0.1 mol / L boric acid-borax buffer (pH 8.3, containing 0.3 mol / L NaCl) to prepare a sample solution with a concentration of 1 mg / mL. According to the above "1. Determination of ACE inhibitory activity", the ACE inhibition rates of the C1, C2 and C3 components were 47.92%, 57.43% and 63.85%, respectively, and the C3 component with the lowest molecular weight showed the best ACE inhibition rate.

[0088] The lyophilized powder of the C3 component obtained by ultrafiltration was prepared into a 30 mg / mL polypeptide solution, and the cicada pupa ACE inhibitory peptide was separated by semi-preparative reversed-phase high performance liquid chromatography (prep-HPLC). Chromatographic conditions: Waters XBridge PrepC18 column (20mm×250mm, 10μm); flow rate: 10mL / min; detection wavelength: 220nm; mobile phase: A is an aqueous solution containing 0.1% TFA (v / v), B is acetonitrile containing 0.1% TFA (v / v); injection volume: 1000μL; elution conditions (expressed by the content of mobile phase A): 0-5min, 95%, 5-20min, 95%-70%, 20-30min, 70%-10%, 30-40min, 10%-95%, 40-45min, 95%; collect components C3-1, C3-2, C3-3, C3-4, C3-5, C3-6, C3-7, C3-8, C3-9 ( Figure 1 A), freeze-dried them respectively, dissolved them in 0.1 mol / L boric acid-borax buffer (pH 8.3, containing 0.3 mol / L NaCl) to prepare a sample solution with a concentration of 1 mg / mL. The ACE inhibition rate of each component was determined according to the above "1. Determination of ACE inhibitory activity".

[0089] The inhibition rate results are shown in Figure 1 B. The C3-8 component had the highest ACE inhibition rate, which was 74.16% at 1 mg / mL. Component C3-8 was selected for subsequent peptide sequence identification.

[0090] 3. Mass spectrometry identification of active peptides

[0091] The C3-8 component obtained in step 2 above was first dissolved in a 0.1% (v / v) trifluoroacetic acid aqueous solution, and then desalted using a C18 column. The column was first eluted with 100% acetonitrile, then balanced with a 0.1% (v / v) trifluoroacetic acid aqueous solution, and after adding the polypeptide sample solution, salt and other impurities were eluted with a 0.1% (v / v) trifluoroacetic acid aqueous solution, and finally the polypeptide was eluted from the column with a 0.1% (v / v) trifluoroacetic acid aqueous solution containing 50% (v / v) acetonitrile and 0.1% (v / v) trifluoroacetic acid aqueous solution, and freeze-dried overnight to obtain a lyophilized powder of the C3-8 component, which was identified using nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS).

[0092] 5 mg of C3-8 component lyophilized powder was dissolved in 100 μL mobile phase A (containing 0.1% formic acid-water solution), the injection volume was 3 μL, the flow rate was 10 μL / min, and the sample was captured on the capture column for 3 minutes, followed by gradient elution chromatography separation on the nanoliter analytical column, and the data-dependent mode was used to automatically switch between MS and MS / MS acquisition. The full scan MS used Orbitrap for primary scanning, with a scanning range of m / z 150-1600 and a resolution set to 120,000. The raw data obtained was processed and analyzed using PEAKS software. ALC (%) stands for average local confidence, which is the confidence of the results of de novo sequencing data. Generally, it is more reliable when it is greater than 80%, and it is very reliable when it is greater than 95%. Peptide Ranker scores were performed on 2627 peptides with ALC scores exceeding 95%, and the score threshold was set to 0.8, from which 161 bioactive peptides with high potential were screened. These 161 peptides were compared with the ACE inhibitory peptide structures in the BIOPEP database, and finally 139 peptides with possible ACE inhibitory activity were screened out.

[0093] The safety, water solubility and absorbability of active peptides have an important impact on their application value. Therefore, ToxinPred, INNOVAGEN, Allergen FP and ADMET Lab 3.0 tools were used to predict the toxicity, solubility, sensitization and absorption characteristics of the above 139 peptides in the human intestine. Finally, Ser-Tyr-Arg-Phe (SYRF), Gly-Pro-Lys-Leu-Phe (GPKLF), Ser-Pro-Arg-Pro-Trp (SPRPW) and Phe-Arg-Gly-Phe (FRGF) were screened out, a total of 4 short peptides with excellent characteristics for subsequent research. The mass spectra of SYRF, GPKLF, SPRPW and FRGF are shown in the figure below. Figure 2 shown.

[0094] Example 2 Molecular docking simulation of the binding of active peptides to ACE

[0095] The ACE protein structure was selected from 1O8A.pdb in the PDB database, and the original ligands on the ACE protein molecule were removed using PyMOL v2.3.2 software (DeLanoScientific LLC, USA), retaining the Zn 2+ Molecular docking was performed using AutoDock 1.5.6 software and visualization was performed using PyMOL v2.3.2 software.

[0096] The docking results showed that all four peptides could be successfully docked with ACE, and all four peptide ligands could form hydrogen bonds with ACE molecules. There were van der Waals forces between Ser-Tyr-Arg-Phe (SYRF), Gly-Pro-Lys-Leu-Phe (GPKLF) and Phe-Arg-Gly-Phe (FRGF) and ACE molecules, salt bridges between Ser-Pro-Arg-Pro-Trp (SPRPW) and Phe-Arg-Gly-Phe (FRGF) and ACE molecules, and π-π stacking between Gly-Pro-Lys-Leu-Phe (GPKLF) and ACE molecules. Table 1 shows the residues, types of interactions and docking energies of the four peptides interacting with ACE. Figure 3 The global image and detailed images of the peptide docking with ACE are shown.

[0097] Table 1 Residues interacting with active peptides and ACE

[0098]

[0099] Example 3 Half Inhibitory Concentration of Active Peptides on ACE

[0100] Four short peptides, Ser-Tyr-Arg-Phe (SYRF), Gly-Pro-Lys-Leu-Phe (GPKLF), Ser-Pro-Arg-Pro-Trp (SPRPW) and Phe-Arg-Gly-Phe (FRGF), were synthesized by Gill Biochemical (Shanghai) Co., Ltd.

[0101] Ser-Tyr-Arg-Phe (SYRF), Gly-Pro-Lys-Leu-Phe (GPKLF), Ser-Pro-Arg-Pro-Trp (SPRPW) or Phe-Arg-Gly-Phe (FRGF) polypeptide solutions at concentrations of 1 mg / mL, 500 μg / mL, 250 μg / mL, 50 μg / mL, 10 μg / mL, and 2 μg / mL were prepared with 0.1 mol / L boric acid-borax buffer (pH 8.3, containing 0.3 mol / L NaCl), and the ACE inhibition rates of polypeptides at different concentrations were detected with reference to the above “1. Determination of ACE inhibitory activity”. The results obtained by fitting analysis using SPSS software are shown in Table 2. The IC values ​​of Ser-Tyr-Arg-Phe (SYRF), Gly-Pro-Lys-Leu-Phe (GPKLF), Ser-Pro-Arg-Pro-Trp (SPRPW) and Phe-Arg-Gly-Phe (FRGF) 50 The values ​​were 0.026 mg / mL, 0.118 mg / mL, 0.34 mg / mL and 1.716 mg / mL respectively.

[0102] Table 2 IC of active peptides against ACE inhibition 50

[0103]

[0104] Note: a, b, c, d represent significant differences among different samples.

[0105] Example 4 Kinetic analysis of the inhibitory effect of active peptides on ACE

[0106] 20 μL of peptide solutions (Ser-Tyr-Arg-Phe (SYRF), Gly-Pro-Lys-Leu-Phe (GPKLF), Ser-Pro-Arg-Pro-Trp (SPRPW) and Phe-Arg-Gly-Phe (FRGF)) of different concentrations (0, 0.1, 0.5 mg / mL) were mixed with 20 μL of 50 mU / mL ACE and incubated at 37°C for 10 min. Then, 40 μL of HHL of different concentrations (0.076, 1.52, 3.04, 3.8, 7.6 mM / L) were added and incubated at 37°C for 40 min. Finally, 50 μL of 1 M HCl was added to terminate the reaction.

[0107] Hippuric acid standard solutions with concentrations of 0.005 mg / mL, 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL and 1 mg / mL were prepared with methanol solution, and the test was performed according to the liquid phase conditions of "1. Determination of ACE inhibitory activity". The relationship between peak area and hippuric acid concentration was plotted to obtain a hippuric acid standard curve of y=247.99x+1.5095, R 2 =0.9991. The amount of hippuric acid generated was calculated based on the hippuric acid standard curve, and the reaction rate v was obtained by the ratio of the amount of hippuric acid generated and the reaction time. The Lineweaver-Burk double reciprocal plot was drawn based on the relationship between the reciprocal of the enzymatic reaction rate (1 / [v]) and the reciprocal of the substrate concentration (1 / [s]) to study the inhibition mode of the cicada pupa protein ACE inhibitory peptide. Figure 4 As shown, FRGF exhibited non-competitive inhibition, SPRPW and GPKLF exhibited mixed inhibition, and SYRF exhibited competitive inhibition.

[0108] Example 5 Study on the thermodynamic parameters of ACE binding active peptides (ITC)

[0109] VP-ITC was used to determine the thermodynamic parameters of the binding of four cicada pupa ACE inhibitory peptides to ACE.

[0110] 0.5 μM ACE solution: 0.5 μM ACE solution was prepared using 0.1 mol / L borate buffer (pH 8.3, containing 0.3 mol / L NaCl).

[0111] 1.9 mM FRGF solution: A 1.9 mM FRGF solution was prepared using 0.1 mol / L borate buffer (pH 8.3, containing 0.3 mol / L NaCl).

[0112] 1.56 mM SPRPW solution: Use 0.1 mol / L borate buffer (pH 8.3, containing 0.3 mol / L NaCl) to prepare a 1.56 mM SPRPW solution.

[0113] 1.79 mM GPKLF solution: A 1.79 mM GPKLF solution was prepared using 0.1 mol / L borate buffer (pH 8.3, containing 0.3 mol / L NaCl).

[0114] 1.75 mM SYRF solution: A 1.75 mM SYRF solution was prepared using 0.1 mol / L borate buffer (pH 8.3, containing 0.3 mol / L NaCl).

[0115] All the above sample solutions were degassed by ultrasonic treatment for 10 min, 1400 μL of ultrapure water was injected into the reference cell, 1400 μL of 0.5 μM ACE solution was injected into the sample cell, and 300 μL of ACE inhibitory peptide solutions of various concentrations were added to the injection needle. The temperature was 37 °C. After the calorimeter reached equilibrium, ACE inhibitory peptide was automatically added, 28 drops were added continuously, each drop was 10 μL, the interval time was 240 s, the stirring speed was 300 rpm, and the calorimetric curve was recorded.

[0116] Microcal Analysis Launcher software was used to analyze the thermodynamic parameters of the thermodynamic reaction of the interaction between ACE inhibitory peptide and ACE. The results are shown in Table 3 and Figure 5 (Thermal flow diagram and fitting curve of peptide binding to ACE) shown.

[0117] Table 3 Thermodynamic parameters of active peptide binding to ACE

[0118]

[0119] The results are as follows:

[0120] The stoichiometric ratio (n) reflects the number of binding sites between ACE inhibitory peptides and ACE. As shown in Table 3, the n values ​​of FRGF, SPRPW and GPKLF are much greater than 1, indicating that the binding of these three ACE inhibitory peptides to ACE is multi-site binding; the n value of SYRF is closer to 1, indicating that it is more likely to be single-site binding. The results of the ITC experiment show that FRGF, SPRPW and GPKLF are more likely to bind to the inactive site of ACE to exert an inhibitory effect, while SYRF is more likely to bind to the active site of ACE inhibitory peptides to exert an inhibitory effect, which is consistent with the inhibition kinetic results.

[0121] The binding constant (Ka) reflects the binding ability of the inhibitory peptide to ACE, and the larger the value, the stronger the binding ability. Among the four peptides, SYRF has the highest Ka value, indicating that its binding to ACE is stronger. As shown in Table 3, the enthalpy change ΔH<0 indicates that the binding of ACE inhibitory peptide to ACE is an exothermic reaction, and the higher the absolute value of ΔH, the more heat is released, which is more conducive to the binding of peptide to ACE. The change in Gibbs free energy ΔG<0 indicates that its binding reaction is spontaneous. The entropy change ΔS>0 indicates that the binding of the inhibitory peptide to ACE will not automatically dissociate, which is in line with the principle of entropy increase. Under the same Gibbs free energy, the negative enthalpy change and positive entropy change have stronger binding driving force and are more conducive to the formation of the complex.

[0122] Example 6 Effect of active peptides on the secretion of NO and ET-1 by HUVEC cells

[0123] HUVEC cell culture: cells were grown in endothelial cell basal medium, and the incubator conditions were 37°C, 5% CO2, and 90% relative humidity. When the cell density reached 80%-90%, the original medium was discarded, 1 mL of trypsin was added to the culture flask and incubated for 2 min to digest the cells, and 3 mL of endothelial cell basal medium was added to terminate the digestion. Carefully blow the cells to make them fall off, transfer the culture medium to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 3 min at 4°C, discard the culture medium, add endothelial cell basal medium to resuspend the cells, and then pass them at a ratio of 1:3. When the color of the culture medium changes from red to yellow, the culture medium needs to be replaced, and the experimental treatment is carried out when the cell density reaches 80%.

[0124] Endothelial cell basal medium was used to prepare Ser-Tyr-Arg-Phe (SYRF) or Gly-Pro-Lys-Leu-Phe (GPKLF) solutions at concentrations of 200 μg / mL, 400 μg / mL, and 800 μg / mL.

[0125] Preparation of Captopril solution: 100 μg / mL Captopril solution (purchased from Beijing Solebow Technology Co., Ltd., catalog number C7510) was prepared using endothelial cell basal culture medium.

[0126] The cell experiments are as follows:

[0127] (1) Control group (blank group): The cultured cells were resuspended and diluted with endothelial cell basal medium at 1×10 5 The cells were inoculated into a 24-well culture plate at a density of 1000 μL cells / mL for culture. Three parallels were set up for each group. The cells were cultured at 37°C for 24 h. The original culture medium was removed, and then 1000 μL of endothelial cell basal culture medium was added to the wells and cultured at 37°C for 24 h.

[0128] (2) SYRF group: The cultured cells were resuspended and diluted with endothelial cell basal medium at 1×10 5 The cells were inoculated at a density of 10 cells / mL into a 24-well culture plate for culture with an inoculation volume of 1000 μL. Three parallels were set up for each group and cultured at 37°C for 24 h. The original culture medium was aspirated, and then 1000 μL of 200 μg / mL (low-dose group), 400 μg / mL (medium-dose group) or 800 μg / mL (high-dose group) Ser-Tyr-Arg-Phe (SYRF) solution was added to the wells and cultured at 37°C for 24 h.

[0129] (3) GPKLF group: The cultured cells were resuspended and diluted with endothelial cell basal medium at 1×10 5The cells were inoculated at a density of 10 cells / mL into a 24-well culture plate for culture with an inoculation volume of 1000 μL. Three parallels were set up for each group and cultured at 37°C for 24 h. The original culture medium was aspirated, and then 1000 μL of 200 μg / mL (low-dose group), 400 μg / mL (medium-dose group) or 800 μg / mL (high-dose group) Gly-Pro-Lys-Leu-Phe (GPKLF) solution was added to the wells and cultured at 37°C for 24 h.

[0130] (4) Captopril group (positive drug group): The cultured cells were resuspended and diluted with endothelial cell basal medium and then diluted to 1×10 5 The cells were inoculated into a 24-well culture plate at a density of 10 cells / mL for culture. The inoculation volume was 1000 μL. Three parallels were set up for each group. The cells were cultured at 37°C for 24 h. The original culture medium was removed, and then 1000 μL of captopril solution was added to a final concentration of 100 μg / mL.

[0131] The NO and ET-1 contents were determined according to the requirements of the NO kit and ET-1 kit (purchased from Nanjing Jiancheng Bioengineering Institute).

[0132] The results are shown in Table 4 and Figure 6 As shown, compared with the blank control group, the NO level of cells treated with peptides was significantly increased, and ET-1 was significantly decreased (p<0.05), and they were concentration-dependent.

[0133] Table 4 Effects of active peptides on the secretion of NO and ET-1 by HUVEC cells

[0134]

[0135]

[0136] Example 7 Effect of active peptides on the proliferation rate of HUVEC cells (cytotoxicity experiment)

[0137] The cell culture conditions are as shown in "Example 5 HUVEC cell culture".

[0138] Use endothelial cell basal medium to prepare Ser-Tyr-Arg-Phe (SYRF) or Gly-Pro-Lys-Leu-Phe (GPKLF) solutions with concentrations of 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, and 800 μg / mL.

[0139] The cell experiments are as follows:

[0140] (1) Control group (blank group): The cultured cells were resuspended and diluted with endothelial cell basal medium and then diluted to 5×10 4The cells were inoculated into a 96-well culture plate at a density of 100 cells / mL for culture. The inoculation volume was 100 μL. Five parallels were set up for each group. The cells were cultured at 37°C for 24 h. The original culture medium was removed, and then 100 μL of endothelial cell basal culture medium was added to the wells and cultured at 37°C for 24 h.

[0141] (2) SYRF group: The cultured cells were resuspended and diluted with endothelial cell basal medium and then diluted to 5×10 4 The cells were inoculated at a density of 10 cells / mL into a 96-well culture plate for culture with an inoculation volume of 100 μL. Five parallels were set up for each group and cultured at 37°C for 24 h. The original culture medium was removed, and then 100 μL of 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, or 800 μg / mL Ser-Tyr-Arg-Phe (SYRF) solution was added to the wells, respectively, and cultured at 37°C for 24 h.

[0142] (3) GPKLF group: The cultured cells were resuspended and diluted with endothelial cell basal medium and then diluted to 5×10 4 The cells were inoculated into 96-well culture plates at a density of 100 cells / mL in an inoculation volume of 100 μL. Five parallels were set up in each group and cultured at 37°C for 24 h. The original culture medium was aspirated, and then 100 μL of 100 μg / mL, 200 μg / mL, 400 μg / mL, 600 μg / mL, or 800 μg / mL Gly-Pro-Lys-Leu-Phe (GPKLF) solution was added to the wells, respectively, and cultured at 37°C for 24 h.

[0143] (4) Captopril group (positive drug group): The cultured cells were resuspended and diluted with endothelial cell basal medium to a concentration of 5×10 4 The cells were inoculated into a 96-well culture plate at a density of 100 cells / mL for culture. The inoculation volume was 100 μL. Five parallels were set up in each group and cultured at 37°C for 24 h. The original culture medium was removed, and then 100 μL of captopril (purchased from Beijing Solebow Technology Co., Ltd., catalog number C7510) was added to a final concentration of 100 μg / mL.

[0144] The cell survival rate was determined according to the MTT kit (purchased from Nanjing Jiancheng Bioengineering Institute).

[0145] The results are shown in Table 5 and Figure 7 As shown, within the range of 100 μg / mL-800 μg / mL, the survival rates of Ser-Tyr-Arg-Phe (SYRF) and Gly-Pro-Lys-Leu-Phe (GPKLF) on HUVEC cells were both above 95%, indicating that they were non-toxic to cells and highly safe.

[0146] Table 5 Effects of active peptides on HUVEC cell survival rate

[0147]

[0148]

[0149] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A polypeptide having ACE inhibitory activity, characterized in that: The amino acid sequence of the polypeptide is Ser-Tyr-Arg-Phe, Gly-Pro-Lys-Leu-Phe, Ser-Pro-Arg-Pro-Trp or Phe-Arg-Gly-Phe.

2. A product, characterized in that: The product comprises an effective dose of at least one of the polypeptides of claim 1; Preferably, the product is food, medicine, health product or nutritional product.

3. The product according to claim 2, characterized in that The drug also contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners may be added to the composition; Preferably, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form; Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions; Preferably, the injection dosage form includes but is not limited to injection solution and injection powder; Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

4. The product according to claim 2, characterized in that The food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; Preferably, the food also includes special dietary food; Preferably, the health care product further contains acceptable excipients.

5. Use of one or more of the polypeptides according to claim 1 in the preparation of products for inhibiting ACE activity, lowering blood pressure, preventing or treating hypertension, heart disease or cardiovascular disease.

6. The use according to claim 5, characterized in that: The product is food, medicine, health product or nutritional product.

7. The use according to claim 5, characterized in that: The drug also contains pharmaceutically acceptable excipients; the pharmaceutical excipients refer to conventional drug carriers in the pharmaceutical field; Preferably, the auxiliary materials include one or more of the following: adhesives such as cellulose derivatives, alginate, gelatin and polyvinyl pyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; wetting agents such as glycerol; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinyl pyrrolidone and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, sorbitan fatty acid and glycerol fatty acid esters; colorants such as titanium dioxide, sunset yellow, methylene blue, medicinal iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate; in addition, other auxiliary agents such as flavoring agents and sweeteners may be added to the composition; Preferably, the dosage form of the drug includes but is not limited to oral dosage form, injection dosage form, and inhalation dosage form; Preferably, the oral dosage form includes but is not limited to tablets, capsules, granules, oral liquids, oral suspensions; Preferably, the injection dosage form includes but is not limited to injection solution and injection powder; Preferably, the inhalation dosage form includes but is not limited to aerosols and powder sprays.

8. The use according to claim 5, characterized in that: The food includes but is not limited to cereal products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages; the food also includes special dietary foods; The health care product also contains acceptable auxiliary materials.

9. A method for preparing the product according to any one of claims 2 to 4, characterized in that: The method comprises mixing at least one of the polypeptides described in claim 1 with at least one acceptable excipient; Preferably, the product is food, medicine, health product or nutritional product.

10. An expression vector or a recombinant microorganism, characterized in that: The expression vector or recombinant microorganism comprises at least one of the polypeptides according to claim 1; Preferably, the vector is selected from a DNA vector, an RNA vector, a plasmid, a transposon vector, a CRISPR / Cas9 vector, or a viral vector; Preferably, the recombinant microorganism is a bacterium or a fungus.

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