Natural royal jelly active peptide with antihypertensive activity as well as preparation and application of natural royal jelly active peptide

Abstract

The invention discloses a natural royal jelly active peptide with antihypertensive activity as well as preparation and application thereof. The preparation method comprises the following steps: removing macromolecular protein of royal jelly by using ethanol precipitation, and removing saccharides and salts by using a Sep-Pak Vac C18 column assisted by elution with 40% acetonitrile. Carrying out rotary evaporation concentration and freeze-drying treatment, extracting with trichloromethane to remove fatty acid, and carrying out ultrafiltration to intercept 1t; therefore, the requirements of mass spectrum on-machine detection can be met. The preparation method provided by the invention is a first attempt for extracting the natural active peptide from the royal jelly, and the royal jelly active peptide extracted by using the method has the advantages of few impurities, high purity, less interference on a mass spectrum identification result and the like. Three royal jelly natural peptides with antihypertensive activity are obtained by screening polypeptide information prepared and detected through the method in a molecular docking mode, the amino acid sequences of the royal jelly natural peptides are PPPPPP, LPPIPS and YNVGPL, and the royal jelly natural peptides have important significance in development of royal jelly active peptide products.

Description

Technical Field

[0001] The invention belongs to the field of active peptide extraction and relates to a natural royal jelly active peptide with blood pressure lowering activity and its preparation and application. Background Art

[0002] Royal jelly is a milky white, light yellow or light orange pulpy substance secreted by the mandibular gland and hypopharyngeal gland of worker bees, which is mainly used to feed queen bees and bee larvae. Royal jelly is rich in a variety of active ingredients, including proteins and peptides, fatty acids, amino acids, vitamins and carbohydrates. Protein is the most active ingredient in royal jelly, with a content of about 12% to 15% in royal jelly, accounting for about 50% of the dry matter content, and is the main source of royal jelly peptides. In addition, there are a small amount of natural peptides in royal jelly, which have been shown to have general antibacterial activity. At present, the information on endogenous peptides in royal jelly is not comprehensive. The royal jelly peptide spectrum results obtained by mass spectrometry identification are of great significance to enrich the royal jelly protein information and strengthen the development of functional royal jelly active peptides.

[0003] At present, royal jelly polypeptides are mainly extracted by enzymatic hydrolysis of royal jelly proteins, and are mostly used for proteomics. There is no method to directly extract polypeptides from royal jelly and use them for peptidomics. Royal jelly has a complex composition, among which high abundance of proteins, sugars and fatty acids bring great interference to the direct extraction of polypeptides. This method effectively removes the above components through ethanol precipitation, C18 column elution and chloroform extraction. The obtained polypeptide extract can meet the requirements of mass spectrometry, and largely avoids the influence on the mass spectrometry identification results.

[0004] Hypertension is a phenomenon in which the pressure value on the blood vessel wall caused by blood flow is continuously higher than normal. It is a chronic disease with high incidence in my country. At present, nearly 245 million people in my country suffer from hypertension, and the adult hypertension prevalence rate is 23.2%. Angiotensin-converting enzyme (ACE) is a key enzyme in the renin-angiotensin-aldosterone system. ACE can directly lead to increased blood pressure by promoting the conversion of angiotensin to angiotensin II, thereby promoting vasoconstriction. Bradykinin can release vasodilating factors to counteract angiotensin II and reduce blood pressure, while ACE has the function of inactivating bradykinin, which can indirectly lead to increased blood pressure. ACE inhibitory peptides are a class of active peptides that can inhibit ACE activity and then inhibit angiotensin II, ultimately achieving the purpose of lowering blood pressure. In recent years, food-derived ACE inhibitory peptides have become a research hotspot in the fields of medicine and food due to their advantages such as natural, safe, small molecular weight and no side effects. Summary of the invention

[0005] The invention aims to provide a natural royal jelly active peptide with blood pressure lowering activity, comprising three natural royal jelly active peptides, whose amino acid sequences are PPPPPP, LPPIPS and YNVGPL respectively.

[0006] The method for preparing a natural royal jelly active peptide having blood pressure lowering activity described in the present invention is achieved by the following steps: (1) Weigh a royal jelly sample, mix the royal jelly with ethanol in a solid-liquid ratio of 1:2, stir thoroughly, and then precipitate overnight at 4°C; (2) Stir the liquid obtained in (1) evenly, transfer it to an EP tube, centrifuge it and collect the supernatant; (3) The supernatant obtained in (2) was transferred to a distillation flask and subjected to rotary evaporation to remove ethanol from the supernatant; (4) The concentrated solution obtained in (3) was filtered through a PES membrane and then the concentrated solution obtained after rotary evaporation was transferred to a new EP tube, and deionized water was added to make the volume to 40 mL to obtain a crude peptide extract; (5) injecting the crude peptide extract obtained in (4) into a Sep-Pak Vac C18 column, eluting with 40% acetonitrile, and collecting the obtained eluate; (6) The solution obtained in (5) was transferred to a distillation flask and rotary evaporated to remove acetonitrile. The concentrated solution obtained by rotary evaporation was transferred to an EP tube and fixed to 40 mL with deionized water to obtain a purified peptide extract; (7) freeze-drying the extract obtained in (6), and collecting the resulting dry matter; (8) The dried product obtained in (7) was prepared into a 5 mg / mL solution using deionized water in a 15 mL centrifuge tube, and an equal volume of chloroform was added. After precipitation on ice for 1 h, the upper liquid was recovered; (9) adding the liquid obtained in (8) into an ultrafiltration tube for centrifugation, collecting the obtained filtrate, and using it for LC-MS / MS detection; (10) The filtrate obtained in (9) was injected into the LC-MS / MS system, and the LC-MS / MS detection results were analyzed using software to obtain peptide sequence data in the sample. Three royal jelly natural peptides with antihypertensive activity were obtained, and their amino acid sequences were PPPPPP, LPPIPS and YNVGPL.

[0007] The mass of the royal jelly sample mentioned in step (1) is 100 g, the volume of ethanol is 200 mL, and the alcohol precipitation time is more than 12 h.

[0008] The centrifugation conditions mentioned in step (2) are 10000 g, 4° C., 45 min.

[0009] The rotary evaporation conditions mentioned in step (3) are 40° C., 25 min.

[0010] The specification of the PES membrane mentioned in step (4) is 0.22 um.

[0011] The elution process mentioned in step (5) is as follows: (1) Activation: Soak the C18 column in 100% acetonitrile in advance for activation; (2) Cleaning: Fill the C18 column with 100% acetonitrile, collect the waste liquid, and repeat this step three times; (3) Sample addition: Add the solution obtained in (2) to the C18 column. After the liquid level drops below the height of the gel column, fill it up with pure water, collect the waste liquid flowing out, and repeat this step three times; (4) Extraction: Add 40% acetonitrile to the C18 column, collect the effluent extract, and repeat this step three times; Cleaning: Fill the C18 column with 100% acetonitrile, collect the waste liquid, and repeat this step three times; (5) Sample addition: Repeatedly add crude peptide extract until the extract is exhausted.

[0012] The rotary evaporation conditions mentioned in step (6) are 40° C., 15 min.

[0013] The volume of the chloroform solution mentioned in step (8) is 2 mL.

[0014] The ultrafiltration tube specification mentioned in step (9) is 3 kDa, and the centrifugation conditions are 5000 g, 4° C., and 45 min.

[0015] For the LC-MS / MS operation process in step (10), a PepMap RSLC analytical column (75 μm x 25 cm) and a PepMap 100 analytical column (75 μm x 2 cm) were used, mobile phase A was pure water, and mobile phase B was 80% acetonitrile solution. The elution conditions were: 0-10 min, 1-6% B, 0.5 mL / min; 10-40 min, 6-22% B, 0.3 mL / min; 40-70 min, 22-60% B, 0.3 mL / min; 70-82 min, 60-90% B, 0.3 mL / min; 82-90 min, 99% B, 0.3 mL / min; mass spectrometry conditions: nano ESI mode was used, the emitter spray voltage was 2 kV, and the scanning range was 300-2000 m / z.

[0016] For the data analysis process mentioned in step (10), the analysis software was Proteome Discover 3.1, the Uniport protein database was used for data retrieval, and the screening criterion for peptide sites was PSM FDR ≤ 0.01.

[0017] Another object of the present invention is to provide the use of the natural royal jelly active peptide in the preparation of antihypertensive drugs or auxiliary antihypertensive health products. Studies have shown that natural royal jelly polypeptides PPPPPP, LPPIPS and YNVGPL have ACE protein binding ability and have antihypertensive activity. It is of great significance for the development of royal jelly active peptide products.

[0018] The present invention is the first attempt to prepare natural active peptides from royal jelly. The main impurities in royal jelly are removed by alcohol precipitation, C18 column elution, chloroform extraction and other steps, and natural active peptides of royal jelly with high purity can be obtained, which can meet the concentration requirements of LC-MS / MS. The natural royal jelly active peptides prepared by this method contain 3 polypeptides with antihypertensive activity, namely PPPPPP, LPPIPS and YNVGPL. The present invention improves the active function information of royal jelly polypeptides, which is of great significance to strengthening the development of functional royal jelly active peptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the molecular weight distribution of the protein in 5 mg / mL peptide extract.

[0020] Figure 2 This is the mass spectrometry result of royal jelly peptide LC-MS / MS.

[0021] Figure 3 It is the molecular structure of natural royal jelly active peptide PPPPPP.

[0022] Figure 4 It is the molecular structure of natural royal jelly active peptide LPPIPS.

[0023] Figure 5 It is the molecular structure of natural royal jelly active peptide YNVGPL.

[0024] Figure 6 This is a schematic diagram of the binding of natural royal jelly active peptide PPPPPP with ACE protein.

[0025] Figure 7 This is a schematic diagram of the binding of natural royal jelly active peptide LPPIPS and ACE protein.

[0026] Figure 8 Schematic diagram of the binding of natural royal jelly active peptide YNVGPL to ACE protein. Specific implementation methods

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited to the scope of the embodiments.

[0028] Example 1 A method for extracting natural royal jelly active peptides

[0029] The specific steps are as follows: (1) Alcohol precipitation: royal jelly and ethanol were mixed in a ratio of 100 g royal jelly to 200 mL ethanol, magnetically stirred for 15 min, and alcohol precipitation was carried out at 4°C overnight; (2) Centrifugation: The next day, the liquid after alcohol precipitation was stirred evenly, transferred to an EP tube, and centrifuged at 10,000 g at 4°C for 45 min. (3) Concentration: Transfer the supernatant after centrifugation to a distillation flask and perform rotary evaporation at 45°C for 20 min to remove ethanol; (4) Filtration: Filter the concentrated solution obtained after rotary evaporation with a PES membrane, transfer it to a new EP tube, add deionized water to make the volume to 40 mL, and obtain a crude peptide extract; (5) Purification: Fill the pre-activated Sep-Pak Vac C18 with 100% acetonitrile and repeat this step three times. Add the crude peptide extract to the C18 column, and when the liquid level drops below the height of the gel column, fill it with pure water and repeat this step three times. Add 40% acetonitrile to the C18 column, collect the effluent extract, and repeat this step three times. Fill the C18 column with 100% acetonitrile, collect the effluent waste liquid, and repeat this step three times. Repeat adding the crude peptide extract until the extract is exhausted; (6) Concentration: The collected sample solution was transferred to a distillation flask and rotary evaporated at 45°C for 15 min to remove acetonitrile. The concentrated solution obtained by rotary evaporation was transferred to an EP tube and deionized water was added to make the volume to 40 mL to obtain the purified peptide extract; (7) Lyophilization: The collected peptide extract is lyophilized and the dried product obtained by lyophilization is collected; (8) Extraction: The obtained dried product was prepared into a 5 mg / mL solution. 2 mL of the solution was added with an equal volume of chloroform. After precipitation on ice for 1 h, the upper liquid was recovered. (9) Ultrafiltration: Add the collected material into a 3 kDa ultrafiltration tube and centrifuge at 5000 g at 4°C for 15 min. Collect the filtrate and set aside.

[0030] In order to verify the reliability of the extraction method, the extract needs to be tested for purity before being tested on the machine. The test indicators include protein content, 10-HDA content, total sugar content and molecular weight distribution.

[0031] Peptide concentration determination: This method uses the Biyuntian protein concentration kit to detect the peptide extract. Take 5μL of 25mg / mL protein standard and add 245μL of diluent to prepare 250mL of 0.5mg / mL protein standard. Take the required amount of A solution and B solution and mix them thoroughly at a volume of 50:1 for use. Dilute the peptide extract to an appropriate concentration for use. Add 0.5mg / mL protein standard to the holes of a 96-well plate at 0, 1, 2, 4, 8, 12, 16, and 20μL, add deionized water to make up to 20μL, and repeat 3 times for each concentration. Take 20μL of the diluted test solution of each sample and point it into the holes of a 96-well plate, and repeat 3 times for each sample. Add 200μL of BCA working solution to each hole and place it at 37℃ for 20-30min. Use an enzyme reader to measure the absorbance value of each well at A562 wavelength. Calculate the protein concentration of the sample based on the obtained standard curve formula and the sample dilution multiple.

[0032] 10-HDA assay: Pre-analysis treatment: Weigh 100 μL of the extract, add methyl p-hydroxybenzoate as a standard, dilute to 10 mL with ethanol, shake well, and filter through a 0.22 μm membrane for HPLC analysis.

[0033] Chromatographic conditions: Chromatographic column: Sepax HP-C18 coLumn (5μm, 150×4.60mm); mobile phase (CH3OH+0.03moL / LHCL+H2O)=55+10+35; measurement wavelength is 210nm; column temperature is 35℃, injection volume is 4μL, flow rate is 1mL / min, 10-HDA is calculated according to the following formula:

[0034]

[0035] Determination of total sugar content: This method uses the Solebao total sugar content kit to detect the extract. Take 0.01g of glucose and dissolve it in 1mL of water to prepare a 10mg / mL standard solution, and further prepare 0, 0.4, 0.6, 0.8, 1.0, 1.2, 1.6, and 2.0mg / mL glucose solutions. Dilute the extract to an appropriate concentration, take 0.1mL of the diluent, add 0.1mL of reagent one, 0.15mL of distilled water, mix well, and boil in a water bath for 30min. Add 0.1mL of reagent two, vortex to mix, add 0.45mL of distilled water, centrifuge at 8000g, 25℃ for 10min, and take the supernatant for detection. Take 100μL of supernatant and 100μL of reagent three, vortex to mix, boil in a water bath for 10min, add 600μL of distilled water, and prepare 800μL of the test solution. Take 200μL of the test solution and point it into the holes of a 96-well plate, and repeat 3 times for each concentration. The absorbance of each sample at A540 was measured using an enzyme-labeled instrument. ΔA of each sample was calculated as A sample - A blank, a standard curve of ΔA was drawn, and the total sugar content of each sample was calculated based on the standard curve.

[0036] Molecular weight distribution determination: The peptide extract was filtered through a 0.22 μm membrane and set aside. The samples were detected using a Superdex peptide 10 / 300GL column combined with an AKTA protein purification system. Mobile phase: 20% ACN solution, detection wavelength: 215 nm, flow rate: 0.5 mL / min, injection volume: 500 μL, and running time: 60 min. Standards were: aprotinin (6511 Da), bacitracin (1486 Da), glutathione oxidation (612.63 Da), and tyrosine (181.19 Da). The experimental results were analyzed using unicorn 7.0 software.

[0037] Table 1 shows the polypeptide content, 10-HDA content and total sugar content of freeze-dried royal jelly. The experimental results show that the polypeptide content in freeze-dried royal jelly is 16.06%. If it is configured as a 10 mg / mL solution, its concentration is 1.6 mg / mL, which meets the standard for mass spectrometry detection. 10-HDA is the main component of fatty acids in royal jelly, and its content is 3.63%, which is much lower than the polypeptide content and has little impact on mass spectrometry detection. The minimum detection limit of the total sugar concentration detection method used in this method is 0.4 mg / mL. The concentration of the peptide extract configured in the experiment is 10 mg / mL, and the total sugar concentration detected is less than 0.4 mg / mL. Therefore, the total sugar content in freeze-dried royal jelly is less than 4%, which does not affect mass spectrometry detection.

[0038] Table 1 Peptide content 10-HDA content Total sugar content 16.06％ 3.63％ <4％

[0039] Figure 1The molecular weight distribution of proteins in 5mg / mL peptide extract. The experimental results show that the molecular weight of proteins in the peptide extract is distributed in the area less than 1500Da, which is consistent with the molecular weight distribution law of polypeptides. There are signal peaks with intensities of 1027mAU and 2703mAU at 600Da and 180Da, respectively, indicating that the molecular weight of polypeptides extracted by this method is concentrated at 180Da, and a small part is distributed at 600Da.

[0040] The filtrate obtained in (9) was injected into the LC-MS / MS system, and the LC-MS / MS detection results were analyzed using software to obtain peptide sequence data in the sample. The LC-MS / MS operation process was as follows: a PepMap RSLC analytical column (75 μm x25 cm) and a PepMap 100 analytical column (75 μm x 2 cm) were used, mobile phase A was 80% acetonitrile solution, and mobile phase B was pure water. The elution conditions were: 0-10 min, 1-6% B, 0.5 mL / min; 10-40 min, 6-22% B, 0.3 mL / min; 40-70 min, 22-60% B, 0.3 mL / min; 70-82 min, 60-90% B, 0.3 mL / min; 82-90 min, 99% B, 0.3 mL / min; nano Obitrap Eclipse mass spectrometer was used for data acquisition, and the mass spectrometry conditions were: nano ESI mode, emitter spray voltage of 2 kV, and scanning range of 300-2000 m / z. Figure 2 This is the mass spectrometry result of royal jelly peptide LC-MS / MS.

[0041] Example 2 Molecular docking of royal jelly natural peptide and ACE protein

[0042] Proteome Discover 3.1 was used to analyze the peptide information obtained by mass spectrometry identification, and the Uniport protein database was used for data retrieval. The screening criteria for peptide sites were PSM FDR≤0.01, and a total of 75 peptide sequences were screened. The Peptide Ranker software was used to evaluate the biological activity possibility of the 75 peptides, and 10 peptide sequences with scores >0.5 were obtained, indicating that these peptides may have potential biological activity.

[0043] The 10 peptide sequences with scores > 0.5 were molecularly docked with ACE protein by Autodock1.5.7 software, and peptide sequences with strong binding ability to ACE protein were screened. The crystal structure of ACE protein (PDB code: 1o86) was obtained on the RCSB PDB website (https: / / www.rcsb.org / ). The results showed that the three peptide sequences with the lowest binding energy were PPPPP, LPPIPS and YNVGPL, which had the strongest binding ability to ACE protein. The binding energy of peptide PPPPP with ACE protein was -2.82kcal / mol, forming two hydrogen bonds, and the participating residues were Ser516 and Arg522. The binding energy of peptide LPPIPS with ACE protein was -2.82kcal / mol, forming two hydrogen bonds, and the participating residues were Thr371 and Asn167. The binding energy of peptide YNVGPL with ACE protein was -2.95kcal / mol, forming two hydrogen bonds, and the participating residues were Ser516 and Arg522. The binding energies of the three natural royal jelly peptides were all less than -1.2 kcal / mol, so it was believed that the natural royal jelly peptides PPPPPP, LPPIPS and YNVGPL had ACE protein binding ability and had antihypertensive activity. The natural royal jelly peptide-ACE protein conformation was visualized and processed by PyMOL software. Figure 3 , Figure 4 and Figure 5 The molecular structure of PPPPPP, LPPIPS and YNVGPL natural royal jelly active peptides. Figure 6 , Figure 7 and Figure 8 Schematic diagram of the binding of PPPPPP, LPPIPS and YNVGPL natural royal jelly active peptides to ACE protein.

[0044] In summary, the natural royal jelly active peptides obtained by the above preparation method have antihypertensive activity, and the royal jelly active peptides extracted by this method have the advantages of less impurities and high purity. This study is of great significance for enriching the royal jelly peptide spectrum information and developing royal jelly active peptide products.

Claims

1. A natural royal jelly peptide with blood pressure lowering activity, characterized in that: The active peptides are three natural royal jelly peptides, and the amino acid sequences thereof are PPPPPP, LPPIPS and YNVGPL respectively.

2. The method for preparing the natural royal jelly peptide with blood pressure lowering activity according to claim 1, characterized in that: This is accomplished by following these steps: (1) Weigh a royal jelly sample, mix the royal jelly with ethanol in a solid-liquid ratio of 1:2, stir thoroughly, and then precipitate overnight at 4°C; (2) Stir the liquid obtained in (1) evenly, transfer it to an EP tube, centrifuge it and collect the supernatant; (3) The supernatant obtained in (2) was transferred to a distillation flask and subjected to rotary evaporation to remove ethanol from the supernatant; (4) The concentrated solution obtained in (3) was filtered through a PES membrane and then the concentrated solution obtained after rotary evaporation was transferred to a new EP tube, and deionized water was added to make the volume to 40 mL to obtain a crude peptide extract; (5) injecting the crude peptide extract obtained in (4) into a Sep-Pak Vac C18 column, eluting with 40% acetonitrile, and collecting the obtained eluate; (6) The solution obtained in (5) was transferred to a distillation flask and rotary evaporated to remove acetonitrile. The concentrated solution obtained by rotary evaporation was transferred to an EP tube and deionized water was added to make the volume to 40 mL to obtain a purified peptide extract; (7) freeze-drying the extract obtained in (6), and collecting the resulting dry matter; (8) The dried product obtained in (7) was prepared into a 5 mg / mL solution using deionized water in a 15 mL centrifuge tube, and an equal volume of chloroform was added. After precipitation on ice for 1 h, the upper liquid was recovered; (9) The liquid obtained in (8) was added to an ultrafiltration tube for centrifugation, and the filtrate was collected and injected into LC-MS / MS for on-machine detection. The LC-MS / MS detection results were analyzed by software to obtain the peptide sequence data in the sample, and the amino acid sequence was PPPPPP, LPPIPS and YNVGPL.

3. The method according to claim 2, characterized in that The alcohol precipitation time in step (1) is more than 12 hours.

4. The extraction method according to claim 2, characterized in that The elution process mentioned in step (5) is as follows: (1) Activation: Soak the C18 column in 100% acetonitrile in advance for activation; (2) Cleaning: Fill the C18 column with 100% acetonitrile, collect the waste liquid, and repeat this step three times; (3) Sample addition: Add the solution obtained in (2) to the C18 column. After the liquid level drops below the height of the gel column, fill it up with pure water, collect the waste liquid flowing out, and repeat this step three times; (4) Extraction: Add 40% acetonitrile to the C18 column, collect the effluent extract, and repeat this step three times; (5) Cleaning: Fill the C18 column with 100% acetonitrile, collect the waste liquid, and repeat this step three times; (6) Sample addition: Repeatedly add crude peptide extract until the extract is exhausted.

5. The method according to claim 2, characterized in that: The volume of the chloroform solution mentioned in step (8) is 2 mL.

6. The method according to claim 2, characterized in that The LC-MS / MS operation process in step (9) uses a 75 μm x 25 cm PepMap RSLC analytical column and a 75 μm x 2 cm PepMap 100 analytical column, mobile phase A is 80% acetonitrile solution, and mobile phase B is pure water; the elution conditions are: 0-10 min, 1-6% B, 0.5 mL / min; 10~40min, 6~22%B, 0.3mL / min; 40-70 min, 22-60% B, 0.3 mL / min; 70-82 min, 60-90% B, 0.3 mL / min; 82-90 min, 99% B, 0.3 mL / min; Mass spectrometry conditions: nano ESI mode, emitter spray voltage of 2 kV, scanning range of 300-2000 m / z.

7. Use of the natural royal jelly active peptide according to claim 1 in the preparation of blood pressure lowering drugs.

8. Use of the natural royal jelly active peptide according to claim 1 in the preparation of health products for assisting in lowering blood pressure.

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