A polypeptide from pupa of cicada with ACE inhibitory activity and preparation method and application thereof

Abstract

The application discloses a kind of cicada pupa polypeptides with ACE inhibitory activity and its preparation method and application, belong to functional food technical field.The method of the present application uses cicada pupa protein as raw material, after enzyme hydrolysis, ultrafiltration, separation and screening, sequencing, molecular docking verification, four pieces of ACE inhibitory activity cicada pupa protein polypeptide with are obtained, and amino acid sequences are Ser-Tyr-Arg-Phe, Gly-Pro-Lys-Leu-Phe, Ser-Pro-Arg-Pro-Trp and Phe-Arg-Gly-Phe.The polypeptide obtained in the application has very strong ACE inhibitory activity, and has little effect on cell proliferation, low cytotoxicity, high safety, and can be used for the development and preparation of blood pressure lowering functional food or medicine, with good application prospect.

Description

Technical Field

[0001] This invention relates to a cicada pupa polypeptide with ACE inhibitory activity, its preparation method and application, belonging to the field of functional food technology. Background Technology

[0002] Hypertension is a chronic disease characterized by high incidence, high disability rate, and high mortality rate, seriously threatening human life. Various external and internal factors can affect blood pressure changes, but the balance between the pressor system—the renin-angiotensin system (RAS)—and the antihypertensive system—the kallikrein-kinin system (KKS)—is the most important regulatory system. ACE in the RAS system is a significant factor causing elevated blood pressure. Therefore, developing ACE inhibitors by inhibiting ACE activity is an effective approach to treating hypertension. Currently, several angiotensin-converting enzyme inhibitors (ACEIs) (such as captopril and lisinopril) are widely used in the clinical treatment of hypertension. These drugs have significant antihypertensive effects in clinical practice, but they also produce some side effects.

[0003] Currently, dietary ACE-inhibiting peptides are typically obtained from proteins via enzymatic digestion, followed by separation, purification, and mass spectrometry identification to obtain peptides with ACE-inhibiting activity. Previous studies have found that ACE-inhibiting peptides exhibit competitive, non-competitive, and mixed inhibitory types, but peptides with competitive inhibition patterns generally possess stronger ACE-inhibiting activity. Applying ACE-inhibiting peptides to human umbilical vein epithelial cells (HUVECs) revealed that the peptides increased cellular NO secretion and reduced ET-1 production, suggesting that ACE-inhibiting peptides may lower blood pressure through this mechanism.

[0004] Cicada pupae are a typical high-protein insect, with a protein content as high as 72%. Currently, silkworm pupa protein peptides have been proven to have significant bioactivity in antioxidation, uric acid reduction, and anti-inflammation. As a nutrient-rich insect resource, cicada pupae also possess high potential for development and utilization. However, research on the functional activity of cicada pupa proteins is still insufficient and requires further exploration.

[0005] Therefore, functional peptides with ACE-inhibiting 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-inhibiting activity is of great significance for developing safer and more effective drugs or functional foods. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a cicada pupa polypeptide with ACE inhibitory activity, its preparation method, and its applications. Using high-quality insect protein, cicada pupa protein, as raw material, the obtained polypeptide exhibits strong ACE inhibitory activity, minimal impact on cell proliferation, low cytotoxicity, and high safety. It can be used in the development of blood pressure-lowering functional foods or pharmaceuticals, and has broad application prospects.

[0007] This invention provides a cicada pupa protein peptide with ACE inhibitory activity, wherein 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 NO.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-maximal inhibitory concentration (IC50) of Ser-Tyr-Arg-Phe is 0.026 mg / mL; the half-maximal inhibitory concentration (IC50) of Gly-Pro-Lys-Leu-Phe is 0.118 mg / mL; the half-maximal inhibitory concentration (IC50) of Ser-Pro-Arg-Pro-Trp is 0.34 mg / mL; and the half-maximal 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.

[0014] In one embodiment of the present invention, the cicada pupa protein peptide is prepared from cicada pupae.

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

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

[0017] (1) After defatting the cicada pupa powder, NaOH was added for extraction, and the precipitate was discarded by centrifugation to obtain cicada pupa protein alkaline extract; pH was adjusted and the mixture was allowed to stand; the cicada pupa protein precipitate was obtained by centrifugation and freeze-dried; enzyme was added for enzymatic hydrolysis, the enzyme was inactivated, and the precipitate was discarded by centrifugation to obtain cicada pupa protein hydrolysate;

[0018] (2) The pupae protein hydrolysate obtained in step (1) was diluted and ultrafiltered with a 1 kDa ultrafiltration membrane to obtain a component with a density of ≤1 kDa. The component was then freeze-dried to obtain a freeze-dried powder.

[0019] (3) Prepare a solution from the lyophilized powder obtained in step (2), and separate the ACE inhibitory peptide of cicada pupa by high performance liquid chromatography; collect multiple components according to the peak conditions and time, and screen them for ACE inhibitory activity to obtain the component with the strongest ACE inhibitory activity.

[0020] (4) The components obtained in step (3) are identified and screened 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 for NaOH extraction is 50~60 ℃ and the extraction time is 3.0~4.0 h; the ratio of defatted cicada pupa powder to NaOH is 1:(20~25) (w / v).

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

[0023] In one embodiment of the present invention, the conditions for adding enzyme for hydrolysis in step (1) are: temperature 55~60 ℃, pH 9.5~10.0, hydrolysis time 3~4 h; the enzyme used for hydrolysis is alkaline protease, and the amount of enzyme added is 800~1000 U / g; the enzyme inactivation conditions are: 100 ℃ for 10 min.

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

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

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

[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 comprising a therapeutically effective amount of an active ingredient and a pharmaceutically acceptable pharmaceutical excipient; the active ingredient comprising 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 peptides can significantly inhibit ACE activity.

[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 excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch, sucrose, etc.; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch, etc.; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides, etc.; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol, etc.; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate, etc.; and other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0032] The present invention also provides a food, medicine, health product or nutritional product, wherein the food, medicine, health product or nutritional product 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 pharmaceutical product 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 excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also 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 injectable dosage form includes, but is not limited to, injectable liquid and injectable powder.

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

[0040] In one embodiment of the present invention, the food includes, but is not limited to, grain 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 product also 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, pharmaceuticals, health products or nutritional products.

[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 pharmaceutical product 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 excipients include one or more of the following: binders such as cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; diluents such as starch, pregelatinized starch, dextrin, sucrose, lactose, mannitol, etc.; fillers such as starch and sucrose; humectants such as glycerin; disintegrants such as sodium carboxymethyl starch, cross-linked polyvinylpyrrolidone, and dry starch; absorption promoters such as quaternary ammonium compounds; surfactants such as polysorbate, fatty acid sorbitan, and fatty acid glycerides; colorants such as titanium dioxide, sunset yellow, methylene blue, and pharmaceutical iron oxide red, etc.; lubricants such as hydrogenated vegetable oil, talc, and polyethylene glycol; coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, and cellulose acetate; and other excipients such as flavoring agents and sweeteners may also 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 injectable dosage form includes, but is not limited to, injectable liquid and injectable powder.

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

[0050] In one embodiment of the present invention, the food includes, but is not limited to, grain 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 product also contains acceptable excipients.

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

[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 obtained cicada pupa polypeptides with ACE inhibitory activity, specifically the sequences Ser-Tyr-Arg-Phe, Gly-Pro-Lys-Leu-Phe, Ser-Pro-Arg-Pro-Trp and Phe-Arg-Gly-Phe. The structure is novel and easy to prepare. It can be obtained by proteolytic digestion of cicada pupae or by artificial synthesis.

[0056] (2) Experiments show that the four peptides obtained in this invention have strong ACE inhibitory activity; among them, the half-maximal inhibitory concentration (IC50) of Ser-Tyr-Arg-Phe is 0.026 mg / mL; the half-maximal inhibitory concentration (IC50) of Gly-Pro-Lys-Leu-Phe is 0.118 mg / mL; the half-maximal inhibitory concentration (IC50) of Ser-Pro-Arg-Pro-Trp is 0.34 mg / mL; and the half-maximal 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 in this invention have little effect on cell proliferation, low cytotoxicity and high safety.

[0058] (4) The ACE inhibitory peptide of this invention is derived from cicada pupa protein, a high-quality protein source that is high in protein, low in fat, and easily digestible. It possesses antioxidant, blood pressure-lowering, and immunomodulatory biological activities, and is also rich in trace elements. Its production cycle is short, resources are abundant, and it has low allergenicity, making it highly efficient and sustainable. It is an ideal alternative to traditional proteins and has broad market application potential. This invention provides a scientific basis for the application of cicada pupa protein in the food nutrition and health industry. Attached Figure Description

[0059] Figure 1 Here are the high-performance liquid chromatograms and the results of ACE inhibitory activity detection for 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 images show the global molecular docking and crossover details; (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 for titrating ACE with active peptides; (A) FRGF; (B) SPRPW; (C) GPKLF; (D) SYRF;

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

[0065] Figure 7 The graph shows the survival rate of HUVEC cells under different treatment conditions. Detailed Implementation

[0066] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the embodiments are for better explanation of 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. Methods for determining ACE inhibitory activity

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

[0070] Preparation of 7.6 mmol / L HHL (malyureylhistylleucine) solution: Dissolve HHL standard (purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number S23032) in 0.1 mol / L borate-borax buffer (pH 8.3, containing 0.3 mol / L NaCl) to prepare a 7.6 mmol / L HHL solution.

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

[0072] The retention time of hippuric acid peak was determined using 0.5 mg / mL hippuric acid as a standard. A blank control was prepared by reacting the sample solution with 0.1 mol / L boric acid-borax buffer (pH 8.3, containing 0.3 mol / L NaCl).

[0073] The content of hippuric acid (HA) in the reaction system was determined by high performance liquid chromatography (HPLC). A Waters XBridge C18 column (250 mm × 4.6 mm, 5 μm) was used, the detection wavelength was 238 nm, the injection volume was 5 μL, and the flow rate was 0.6 mL / min. Mobile phase A was 10% acetonitrile (0.1% TFA), and mobile phase B was pure acetonitrile. The column temperature was 40 ℃.

[0074] ACE inhibition rate (%) =

[0075] 2. Methods for determining peptide content

[0076] Weigh 500 mg of cicada pupa protein hydrolysate into a 25 mL volumetric flask, and dilute to 25 mL with 15% trichloroacetic acid solution. After centrifugation, collect the supernatant and determine the protein content of the supernatant using the Kjeldahl method according to 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 and dilute to 10 mL with 5% trichloroacetic acid solution. Determine the free amino acid content in the cicada pupa protein hydrolysate according to the method for determining free amino acids in GB 22492-2008 "Soybean Peptide Powder". X 2.

[0077] Peptide content (g / 100g) = X 1 - X 2;

[0078] In the formula X 1 indicates the content of acid-soluble protein, in g / 100g;

[0079] In the formula X 2 indicates the content of free amino acids, in g / 100g.

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

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

[0082] 1. Proteolytic enzyme digestion of cicada pupae

[0083] After defatting, cicada pupa powder was extracted for 3.0 h at a reaction temperature of 60 ℃, a NaOH concentration of 0.075 mol / L, and a material-to-liquid ratio of 1:25 (w / v). The precipitate was discarded by centrifugation at 4000 rpm for 20 min at 4 ℃ to obtain an alkaline extract of cicada pupa protein. The pH of the alkaline extract was adjusted to 4.2 (isoelectric point of cicada pupa protein) with 1 mol / L HCl. The extract was allowed to stand overnight at 4 ℃ and centrifuged at 4000 rpm for 20 min to obtain a cicada pupa protein precipitate. After freeze-drying, cicada pupa protein (CCP) was obtained, with a CCP protein content of 81.8 g / 100 g.

[0084] CCP was enzymatically hydrolyzed at a hydrolysis temperature of 60 ℃, pH 9.5, hydrolysis time of 3 h, substrate concentration (the proportion of protein in the sample to the total mass of the mixture) of 4%, and alkaline protease 6 L (purchased from DuPont, USA) with an enzyme dosage of 1000 U / g. After the reaction, the enzyme was inactivated at 100 ℃ for 10 min, and the precipitate was discarded after centrifugation at 8000 rpm for 20 min at 4 ℃ to obtain the pupal pupa protease hydrolysate (CPPH). According to the above "2. Method for Determination of Peptide Content", the peptide content was determined to be 82.8%.

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

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

[0087] The C1, C2, and C3 fractions obtained by ultrafiltration were prepared into lyophilized powders by rotary evaporation and vacuum freeze-drying. These powders were then dissolved in 0.1 mol / L borate-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-mentioned "1. Determination of ACE Inhibitory Activity", the ACE inhibition rates of fractions C1, C2, and C3 were 47.92%, 57.43%, and 63.85%, respectively. Fraction C3, with the lowest molecular weight, exhibited the best ACE inhibition rate.

[0088] The lyophilized C3 fraction obtained by ultrafiltration was prepared into a 30 mg / mL peptide solution, and the ACE inhibitory peptide of cicada pupa was separated by semi-preparative reversed-phase high-performance liquid chromatography (prep-HPLC). Chromatographic conditions: Waters XBridge Prep C18 column (20 mm × 250 mm, 10 μm); flow rate: 10 mL / min; detection wavelength: 220 nm; mobile phase: A is an aqueous solution containing 0.1% TFA (v / v), B is acetonitrile containing 0.1% TFA (v / v); injection volume: 1000 μL; elution conditions (expressed as the content of mobile phase A): 0-5 min, 95%; 5-20 min, 95%-70%; 20-30 min, 70%-10%; 30-40 min, 10%-95%; 40-45 min, 95%; fractions C3-1, C3-2, C3-3, C3-4, C3-5, C3-6, C3-7, C3-8, and C3-9 were collected according to time. Figure 1 A), the components were lyophilized separately and dissolved in 0.1 mol / L borate-borax buffer (pH 8.3, containing 0.3 mol / L NaCl) to prepare sample solutions with a concentration of 1 mg / mL. The ACE inhibition rate of each component was determined according to the above-mentioned "1. Determination of ACE Inhibitory Activity".

[0089] The inhibition rate results are shown in Figure 1 B, it was found that the C3-8 fraction had the highest ACE inhibition rate, with an ACE inhibition rate of 74.16% at 1 mg / mL. Fraction C3-8 was selected for subsequent peptide sequence identification.

[0090] 3. Mass spectrometry identification of active peptides

[0091] The C3-8 fraction 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 equilibrated with a 0.1% (v / v) trifluoroacetic acid aqueous solution. After adding the peptide sample solution, impurities such as salts were eluted with a 0.1% (v / v) trifluoroacetic acid aqueous solution. Finally, the peptides were eluted from the column with a 0.1% (v / v) trifluoroacetic acid aqueous solution containing 50% (v / v) acetonitrile. The lyophilized C3-8 fraction was obtained overnight and identified using nano-liquid chromatography-tandem mass spectrometry (NanoLC-MS / MS).

[0092] 5 mg of C3-8 fraction lyophilized powder was dissolved in 100 μL of mobile phase A (containing 0.1% formic acid-water solution), with an injection volume of 3 μL and a flow rate of 10 μL / min. The sample was captured on a trap column for 3 minutes, followed by gradient elution chromatography on a nano-scale analytical column. Data acquisition was automatically switched between MS and MS / MS in data-dependent mode. Full-scan MS was performed using Orbitrap for primary scanning, with a scan range of m / z 150-1600 and a resolution of 120,000. The raw data were processed and analyzed using PEAKS software. ALC (%) stands for Average Local Confidence, representing the confidence level of de novo sequencing data. Generally, a score greater than 80% is considered reliable, and a score greater than 95% is highly reliable. Peptide Ranker scoring was performed on 2627 peptides with ALC scores exceeding 95%, with a score threshold of 0.8. From these, 161 peptides with high potential bioactivity were selected. These 161 peptides were compared with the ACE inhibitory peptide structures in the BIOPEP database, and 139 peptides that may have ACE inhibitory activity were finally screened out.

[0093] The safety, water solubility, and absorption of bioactive peptides significantly impact 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 aforementioned 139 peptides in the human intestinal tract. Ultimately, four short peptides with excellent properties—Ser-Tyr-Arg-Phe (SYRF), Gly-Pro-Lys-Leu-Phe (GPKLF), Ser-Pro-Arg-Pro-Trp (SPRPW), and Phe-Arg-Gly-Phe (FRGF)—were selected for further research. The mass spectra of SYRF, GPKLF, SPRPW, and FRGF are shown below. Figure 2 As shown.

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

[0095] The ACE protein structure was selected from the PDB database as 1O8A.pdb. PyMOL v2.3.2 software (DeLanoScientific LLC, USA) was used to remove the original ligands from the ACE protein molecule, retaining 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 successfully dock with ACE, and all four peptide ligands could form hydrogen bonds with the ACE molecule. Ser-Tyr-Arg-Phe (SYRF), Gly-Pro-Lys-Leu-Phe (GPKLF), and Phe-Arg-Gly-Phe (FRGF) exhibited van der Waals forces with the ACE molecule, while Ser-Pro-Arg-Pro-Trp (SPRPW) and Phe-Arg-Gly-Phe (FRGF) formed salt bridges with the ACE molecule, and Gly-Pro-Lys-Leu-Phe (GPKLF) showed π-π stacking with the ACE molecule. Table 1 shows the residues, interaction types, and docking energies of the four peptides interacting with ACE. Figure 3 The images show both global and detailed views of the docking of the peptide with ACE.

[0097] Table 1. Interacting residues of active peptides with ACE

[0098]

[0099] Example 3: Half-maximal inhibitory concentration of active peptides against ACE

[0100] The 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 Jier Biochemical (Shanghai) Co., Ltd.

[0101] Prepare 1 mg / mL, 500 μg / mL, 250 μg / mL, 50 μg / mL, 10 μg / mL, and 2 μg / mL peptide solutions of Ser-Tyr-Arg-Phe (SYRF), Gly-Pro-Lys-Leu-Phe (GPKLF), Ser-Pro-Arg-Pro-Trp (SPRPW), or Phe-Arg-Gly-Phe (FRGF) using 0.1 mol / L borate-borax buffer (pH 8.3, containing 0.3 mol / L NaCl). Detect the ACE inhibition rate of different peptide concentrations according to the above-described "1. Method for Determination of ACE Inhibitory Activity". The results of fitting analysis using SPSS software are shown in Table 2. The IC50 values ​​for Ser-Tyr-Arg-Phe (SYRF), Gly-Pro-Lys-Leu-Phe (GPKLF), Ser-Pro-Arg-Pro-Trp (SPRPW), and Phe-Arg-Gly-Phe (FRGF) are also shown. 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. IC50 of active peptides on ACE inhibition 50

[0103]

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

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

[0106] 20 μL of peptide solutions at different concentrations (0, 0.1, 0.5 mg / mL) (Ser-Tyr-Arg-Phe (SYRF), Gly-Pro-Lys-Leu-Phe (GPKLF), Ser-Pro-Arg-Pro-Trp (SPRPW), and Phe-Arg-Gly-Phe (FRGF) respectively) were mixed with 20 μL of 50 mU / mL ACE and incubated at 37 °C for 10 min. Then, 40 μL of HHL at 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] Standard solutions of hippuric acid 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 using methanol solution. The solutions were analyzed under the liquid chromatography conditions described in "1. Determination of ACE Inhibitory Activity". A standard curve for hippuric acid was obtained by plotting the relationship between peak area and hippuric acid concentration: y = 247.99x + 1.5095, R² = 0.9991. The amount of hippuric acid produced was calculated based on the standard curve, and the reaction rate v was obtained by calculating the ratio of hippuric acid production to reaction time. A Lineweaver-Burk double reciprocal plot was plotted based on the relationship between the reciprocal of the enzyme reaction rate (1 / [v]) and the reciprocal of the substrate concentration (1 / [s]) to investigate the inhibitory mode of the ACE-inhibiting peptide in cicada pupa protein. Figure 4 As shown, FRGF exhibits non-competitive inhibition, SPRPW and GPKLF exhibit mixed inhibition, and SYRF exhibits competitive inhibition.

[0108] Example 5: Thermodynamic parameter study of ACE-bound active peptides (ITC)

[0109] The thermodynamic parameters of the binding of four ACE-inhibiting peptides from cicada pupae to ACE were determined using VP-ITC.

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

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

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

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

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

[0115] All sample solutions were degassed by sonication for 10 min. 1400 μL of ultrapure water was injected into the reference cell, and 1400 μL of 0.5 μM ACE solution was injected into the sample cell. 300 μL of ACE inhibitory peptide solutions of various concentrations were added to the injection needle. The temperature was 37 ℃. After the calorimeter reached equilibrium, ACE inhibitory peptides were automatically added dropwise in 28 drops, 10 μL each, at 240 s intervals. The stirring speed was 300 rpm, and the calorimetric curve was recorded.

[0116] Thermodynamic parameters of the interaction between ACE inhibitory peptides and ACE were analyzed using Microcal Analysis Launcher software. The results are shown in Table 3 and... Figure 5 (The heat flow graph and fitting curve of peptide binding to ACE are shown.)

[0117] Table 3. Thermodynamic parameters of bioactive peptides binding to ACE

[0118]

[0119] The results are as follows:

[0120] The stoichiometric ratio (n) reflects the number of binding sites of the ACE inhibitory peptide to ACE. Table 3 shows that the n values ​​for FRGF, SPRPW, and GPKLF are all much greater than 1, indicating that these three ACE inhibitory peptides bind to ACE at multiple sites; the n value for SYRF is closer to 1, indicating that it is more likely to bind at a single site. ITC experiments show that FRGF, SPRPW, and GPKLF are more likely to bind to the inactive sites of ACE to exert their inhibitory effect, while SYRF is more likely to bind to the active sites of the ACE inhibitory peptide to exert its inhibitory effect, which is consistent with the inhibition kinetics results.

[0121] The binding constant (Ka) reflects the binding ability of the repressor peptide to ACE; the larger the value, the stronger the binding ability. Among the four peptides, SYRF has the highest Ka value, indicating a stronger binding effect with ACE. Table 3 shows that the enthalpy change ΔH < 0, indicating that the binding of the ACE repressor peptide to ACE is an exothermic reaction, and the higher the absolute value of ΔH, the more heat is released, further promoting the binding of the peptide to ACE. The Gibbs free energy change ΔG < 0 indicates that the binding reaction is spontaneous. The entropy change ΔS > 0 indicates that the binding of the repressor peptide to ACE does not involve spontaneous dissociation, conforming to the principle of entropy increase. Under the same Gibbs free energy, a negative enthalpy change and a positive entropy change indicate a stronger driving force for binding, which is more conducive to the formation of the complex.

[0122] Example 6: Effects of bioactive peptides on NO and ET-1 secretion levels in HUVEC cells

[0123] HUVEC cell culture: Cells were grown in endothelial cell basal medium under incubation conditions of 37°C, 5% CO2, and 90% relative humidity. When the cell density reached 80%-90%, the original medium was discarded, and 1 mL of trypsin was added to the culture flask for 2 min to digest the cells. 3 mL of endothelial cell basal medium was then added to stop the digestion. The cells were carefully detached by pipetting, and the medium was transferred to a 15 mL centrifuge tube. The tube was centrifuged at 1000 rpm for 3 min at 4°C, the medium was discarded, and the cells were resuspended in endothelial cell basal medium. The cells were then passaged at a 1:3 ratio. The medium was changed when the color of the culture medium changed from red to yellow. Experimental treatments were performed when the cell density reached 80%.

[0124] 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 were prepared using endothelial cell basal culture medium.

[0125] Preparation of captopril solution: Prepare a 100 μg / mL captopril solution (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number C7510) 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 a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1000 μL per well into 24-well culture plates and cultured in triplicate. The plates were cultured at 37 °C for 24 h. The original culture medium was removed, and then 1000 μL of endothelial cell basal medium was added to each well. The plates were then cultured at 37 °C for 24 h.

[0128] (2) SYRF group: The cultured cells were resuspended and diluted with endothelial cell basal medium at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1000 μL / mL into 24-well plates and cultured in triplicate. After 24 h at 37 °C, the original culture medium was removed, and 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 each well. The plates were then cultured at 37 °C for 24 h.

[0129] (3) GPKLF group: The cultured cells were resuspended and diluted with endothelial cell basal medium and then diluted with 1×10 5Cells were seeded at a density of 1000 μL / mL into 24-well plates and cultured in triplicate. The plates were incubated 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. The plates were then incubated at 37 °C for 24 h.

[0130] (4) Captopril group (positive control group): The cultured cells were resuspended in endothelial cell basal medium and diluted with 1×10⁻⁶ mol / L. 5 The cells were seeded at a density of 1000 μL / mL into 24-well culture plates and cultured in triplicate. The culture was carried out at 37 °C for 24 h. The original culture medium was removed and 1000 μL of captopril solution was added to bring the final concentration to 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 Biotechnology Institute).

[0132] The results are shown in Table 4 and Figure 6 As shown, compared with the blank control group, the cells treated with peptides showed a significant increase in NO and a significant decrease in ET-1. p<0.05 ), and exhibits concentration dependence.

[0133] Table 4. Effects of bioactive peptides on NO and ET-1 secretion levels in HUVEC cells.

[0134]

[0135] Example 7 Effect of bioactive peptides on the proliferation rate of HUVEC cells (cytotoxicity assay)

[0136] Cell culture conditions are as shown in "Example 5 HUVEC Cell Culture".

[0137] 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 were prepared using endothelial cell basal culture medium.

[0138] The cell experiments are as follows:

[0139] (1) Control group (blank group): The cultured cells were resuspended and diluted with endothelial cell basal medium at 5×10⁻⁶. 4Cells were seeded at a density of 100 μL / mL into 96-well culture plates and cultured in 5 replicates per group. The plates were cultured at 37 °C for 24 h. The original culture medium was aspirated, and then 100 μL of endothelial cell basal medium was added to each well. The plates were then cultured at 37 °C for 24 h.

[0140] (2) SYRF group: The cultured cells were resuspended and diluted with endothelial cell basal medium at 5×10⁻⁶ ppm. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates and cultured in 5 replicates per group. After 24 h at 37 °C, 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. The plates were then cultured at 37 °C for 24 h.

[0141] (3) GPKLF group: The cultured cells were resuspended and diluted with endothelial cell basal medium and then diluted with 5×10 4 Cells were seeded at a density of 100 μL / mL into 96-well plates and cultured in 5 replicates per group. After 24 h at 37 °C, 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 Gly-Pro-Lys-Leu-Phe (GPKLF) solution was added to each well. The plates were then cultured at 37 °C for 24 h.

[0142] (4) Captopril group (positive control group): The cultured cells were resuspended and diluted with endothelial cell basal medium and then injected with 5×10⁻⁶ mg / L. 4 Cells were seeded at a density of 100 μL / mL into 96-well plates for culture. Five replicates were set up for each group. The plates were cultured at 37 °C for 24 h. The original culture medium was aspirated, and then 100 μL of captopril (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number C7510) was added to bring the final concentration to 100 μg / mL.

[0143] Cell viability was determined according to the requirements of the MTT kit (purchased from Nanjing Jiancheng Biotechnology Institute).

[0144] The results are shown in Table 5 and Figure 7 As shown, within the range of 100 μg / mL to 800 μg / mL, the survival rate of HUVEC cells by Ser-Tyr-Arg-Phe (SYRF) and Gly-Pro-Lys-Leu-Phe (GPKLF) was above 95%, indicating that they are non-toxic to cells and have high safety.

[0145] Table 5. Effects of bioactive peptides on HUVEC cell survival.

[0146]

[0147] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A polypeptide with 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 contains at least one of the polypeptides of claim 1 in an effective dose; the product is a food, health product, or nutritional product.

3. The product according to claim 2, characterized in that, The food products include grain products, vegetable products, fruit products, meat products, seafood, egg products, dairy products, soy products, and beverages.

4. The product according to claim 2, characterized in that, The food items mentioned also include special dietary foods.

5. The product according to claim 2, characterized in that, The health products also contain acceptable excipients.

6. A method for preparing the product according to any one of claims 2 to 5, characterized in that, The method includes mixing at least one of the polypeptides of claim 1 with at least one acceptable excipient.

7. An expression carrier, characterized in that, The expression vector encodes at least one of the polypeptides of claim 1.

8. The expression vector according to claim 7, characterized in that, The expression vector is selected from DNA vectors, RNA vectors, or viral vectors.

9. The expression vector according to claim 8, characterized in that, The DNA vector is a plasmid, a transposon vector, or a CRISPR / Cas9 vector.

10. A recombinant microorganism, characterized in that, The recombinant microorganism comprises at least one of the polypeptides of claim 1.

11. The recombinant microorganism according to claim 10, characterized in that, The recombined microorganisms are bacteria or fungi.

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