CN115960166B - Selenium-enriched peptide for reducing blood pressure and application thereof - Google Patents
Selenium-enriched peptide for reducing blood pressure and application thereof Download PDFInfo
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- CN115960166B CN115960166B CN202210989287.XA CN202210989287A CN115960166B CN 115960166 B CN115960166 B CN 115960166B CN 202210989287 A CN202210989287 A CN 202210989287A CN 115960166 B CN115960166 B CN 115960166B
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- selenium
- antihypertensive
- ace
- rich peptide
- peptide
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- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 116
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 114
- 239000011669 selenium Substances 0.000 title claims abstract description 114
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 96
- 230000036772 blood pressure Effects 0.000 title claims abstract description 13
- 230000001603 reducing effect Effects 0.000 title description 4
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- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 claims abstract description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims abstract description 4
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a blood pressure lowering selenium-rich peptide and application thereof. The antihypertensive selenium-rich peptide has the amino acid sequence as shown in SEQ ID NO:1, and the sulfur element in methionine M in the antihypertensive selenium-rich peptide is replaced by selenium. The inventor obtains the antihypertensive selenium-rich peptide with higher selenium content from oyster, and the antihypertensive selenium-rich peptide has higher ACE inhibition activity and antihypertensive effect, and defines a specific activity action mechanism, so that the antihypertensive selenium-rich peptide has wide application; meanwhile, the utility value of the oyster is improved, and the processing and the utilization of the oyster can be promoted.
Description
Technical Field
The invention belongs to the technical field of selenium-enriched peptides, and particularly relates to a blood pressure-reducing selenium-enriched peptide and application thereof.
Background
Oyster (Oyster) is the first large cultured shellfish in the world, and the culture yield of Chinese Oyster is more than 80% of the total yield in the world. However, oyster processing utilization is low, about 30% -40%, half of which is frozen processed products, and full utilization of nutritional value and medicinal value is lacking. Oyster is used as a raw material of Chinese traditional medicine and food homologous foods, has rich protein content (the dry basis accounts for 45% -57%), and is a high-quality raw material for preparing bioactive peptides. A great deal of researches show that oyster protein peptide has the functional characteristics of antioxidation, blood pressure reduction, antibiosis, fatigue resistance, immunoregulation, tumor resistance and the like. In addition, oyster is also rich in microelements such as zinc, selenium and the like, wherein the content of selenium is 0.36-1.30mg/kg.
Selenium is a trace element closely related to human health and has important effect on preventing cardiovascular and cerebrovascular diseases. In recent years, organic selenium has been receiving much attention because of its advantages of higher bioactivity and safety, easy absorption by human body, and the like, compared with inorganic selenium. Inorganic selenium can be converted to an organic form in combination with polysaccharides, polypeptides or proteins in a variety of organisms. Bioactive seleno-peptides have become a research hotspot in the field of bioactive peptides as an important source of organic selenium. Liu et al (Liu K,Du R,Chen F.Antioxidant activities of Se-MPS:A selenopeptide identified from selenized brown rice protein hydrolysates[J].LWT-Food Science and Technology,2019,111:555-560.) found that selenium-enriched brown rice protein antioxidant peptide (SeMet-Pro-Ser) exhibited higher ORAC value, ABTS. Cndot.+ scavenging activity and hexavalent chromium reducing activity than Met-Pro-Ser, and selenium exhibited synergistic effect on the antioxidant activity of the peptide. Zhu et al (Zhu J,Du M,Wu M,et al.Preparation,physicochemical characterization and identification of two novel mixed ACE-inhibiting peptides from two distinct tea alkali-soluble protein[J].European Food Research and Technology,2020,246(7):1483-1494.) isolated novel mixed ACE inhibitory peptides Se-TAPep 1 (IC 50 =13.39 mg/mL) and TAPep 1 (IC 50 =23.56 mg/mL) from selenium-rich basic tea protein and common basic tea protein, respectively, and indicated that the ACE inhibitory activity was closely related to selenium content. However, the research on ACE inhibitory activity selenide is focused on the aspects of preparation, separation, purification and the like of selenide, few reports on amino acid sequences are provided, and the research on specific activity action mechanism of the selenide is lacked.
The patent name of the patent is an oyster-derived ACE inhibitory and anti-tumor active peptide, and discloses a small molecule active peptide derived from oyster. The small molecule active peptide has strong ACE inhibitory activity and breast cancer resisting activity, belongs to food-derived antihypertensive peptide, and can be used for treating cardiovascular and cerebrovascular diseases and breast cancer related diseases, in particular for health care and treatment of hypertension. But the small molecule active peptide does not contain selenium.
The research report on selenium peptide of marine animal source, especially oyster source is less, and the development of selenium peptide of oyster source has important research significance and economic value.
Disclosure of Invention
The invention aims to overcome the defect of oyster-derived high-activity selenium peptide and provides a blood pressure-reducing selenium-rich peptide. The inventor obtains the antihypertensive selenium-rich peptide with higher selenium content from oyster, and the antihypertensive selenium-rich peptide has higher ACE inhibition activity and antihypertensive effect, and defines a specific activity action mechanism, so that the antihypertensive selenium-rich peptide has wide application; meanwhile, the utility value of the oyster is improved, and the processing and the utilization of the oyster can be promoted.
The invention also aims to provide the application of the antihypertensive selenium-rich peptide in preparation of antihypertensive drugs.
The invention also aims to provide the application of the antihypertensive selenium-rich peptide in preparing functional foods.
The invention also aims to provide the application of the antihypertensive selenium-rich peptide in preparing health care products.
The invention also aims to provide application of the antihypertensive selenium-rich peptide in preparation of ACE inhibitors.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
A antihypertensive selenium-rich peptide having the amino acid sequence as set forth in seq id no:1 (QAMNEATGGK, i.e. Gln-Ala-Met-Asn-Glu-Ala-Thr-Gly-Gly-Lys), and the sulfur element in methionine M in the antihypertensive selenium-rich peptide is replaced by selenium, and the replaced sequence is recorded as QASEMNEATGGK.
The inventor of the invention utilizes enzymolysis, ultrafiltration separation, two-step RP-HPLC separation and purification, LC-MS/MS peptide spectrum characterization and molecular docking technology screening to obtain a novel antihypertensive selenium-rich peptide QASEMNEATGGK with the molecular weight of 1053.3994Da. The antihypertensive selenium-rich peptide has good antihypertensive effect and wide application.
The research of the invention shows that the antihypertensive selenium-rich peptide can inhibit the secretion of ET-1 and promote the release of NO, and has wide application in preparing antihypertensive drugs.
The invention discloses application of antihypertensive selenium-rich peptide in preparation of antihypertensive drugs.
Preferably, the antihypertensive selenium-rich peptide is applied to preparation of medicines for inhibiting ET-1 secretion.
Preferably, the antihypertensive selenium-rich peptide is applied to the preparation of a medicament for promoting the release of NO.
The application of the antihypertensive selenium-rich peptide in preparing functional food is also within the protection scope of the invention.
The application of the antihypertensive selenium-rich peptide in preparing antihypertensive drugs is also within the protection scope of the invention.
The application of the antihypertensive selenium-rich peptide in preparing ACE inhibitor is also within the protection scope of the invention.
The research shows that the antihypertensive selenium-rich peptide (QASEMNEATGGK) can form a complex with ACE through hydrogen bond, hydrophobic interaction and metal-receptor interaction, thereby effectively inhibiting ACE activity. Specifically: butt-joint of QASEMNEATGGK with ACE hydrogen bonds are formed at several positions Trp59, try62, asn66, asn70, leu139, ala356, his410, glu411 and Pro515, wherein the hydrogen bond action with the acting position of Glu411 can cause distortion of tetrahedrally coordinated Zn 2+ to a certain extent, thereby leading to loss of catalytic activity of ACE. Meanwhile, QASEMNEATGGK can also directly generate metal-acceptor action with the active center Zn701 of ACE to further block the combination of ACE and Zn 2+, thereby inhibiting the catalytic activity of ACE. Furthermore, seMet of QASEMNEATGGK can also form hydrophobic interactions with amino acid residues Val518, pro519 of ACE.
Preferably, the antihypertensive selenium-rich peptide is used for preparing an ACE inhibitor which is combined with amino acid residues Trp59, try62, asn66, asn70, leu139, ala356, his410, glu411 and Pro515 of ACE through hydrogen bonds.
Preferably, the use of the antihypertensive selenium-rich peptide for the preparation of an ACE inhibitor that binds to amino acid residues Val518, pro519 of ACE via hydrophobic interactions.
Preferably, the antihypertensive selenium-rich peptide is applied to the preparation of an ACE inhibitor for inhibiting the combination of ACE and Zn 2+.
Compared with the prior art, the invention has the following beneficial effects:
The inventor obtains the antihypertensive selenium-rich peptide with higher selenium content from oyster, and the antihypertensive selenium-rich peptide has higher ACE inhibition activity and antihypertensive effect, and defines a specific activity action mechanism, so that the antihypertensive selenium-rich peptide has wide application; meanwhile, the utility value of the oyster is improved, and the processing and the utilization of the oyster can be promoted.
Drawings
FIG. 1 is a chromatogram of a primary preparation (FIG. 1A) and a secondary preparation (FIG. 1B).
FIG. 2 shows the molecular docking of antihypertensive selenium-rich peptides with ACE; wherein fig. 2A is an ACE-QASEMNEATGGK complex; fig. 2B is a 2D schematic of the interaction of QASEMNEATGGK with amino acid residues of ACE.
FIG. 3 is an effect of antihypertensive selenium-rich peptides on EA.hy926 cell viability.
FIG. 4 is a graph showing the effect of antihypertensive selenium-rich peptides on NO content in EA.hy926 cells.
FIG. 5 shows the effect of antihypertensive selenium-rich peptides on ET-1 content of EA.hy926 cells.
Detailed Description
The invention is further illustrated below with reference to examples. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental procedures in the examples below, without specific details, are generally performed under conditions conventional in the art or recommended by the manufacturer; the raw materials, reagents and the like used, unless otherwise specified, are those commercially available from conventional markets and the like. Any insubstantial changes and substitutions made by those skilled in the art in light of the above teachings are intended to be within the scope of the invention as claimed.
The main materials and reagents used in each example are described below:
Selenium-enriched oyster protein is provided by the coastal selenium-enriched functional agricultural institute of North Bay; alkaline protease (2X 10 5 U/g), neutral protease (2X 10 5 U/g), papain (2X 10 5 U/g), trypsin (2500 UspU/mg), nanning Pang Bo Bioengineering Co., ltd; angiotensin converting enzyme (derived from rabbit lung), hippocampal-histidyl-leucine (N-hippuryl-His-Leu tetrahydrate, HHL), sigma in the united states; thiazole blue (MTT), shanghai microphone, biochemical technologies limited; DMEM high sugar medium, fetal bovine serum, diabody (penicillin/streptomycin), company Gibico in usa; nitric Oxide (NO) detection kit, biyun biotechnology limited; human endothelin-1 (ET-1) enzyme linked immunosorbent assay kit, CUSABIO technology Co., ltd; the other reagents were all analytically pure.
ACE inhibitory activity in each example was determined as follows:
The sample to be tested was formulated with 0.1mol/L boric acid-borax buffer (pH 8.3, containing 0.3mol/L NaCl) to give an ACE inhibitor (ACEI) solution of the desired concentration. The reaction sample addition sequence is as follows: after adding 10. Mu.L of ACE solution (0.1U/mL), 10. Mu.L of ACEI was added to the sample tube, the mixture was preheated in a 37 ℃ water bath for 5min, then 30. Mu. LHHL solution (5.0 mmol/L) was added, the mixture was reacted in a 37 ℃ water bath for 1h, then 80. Mu.L of HCl solution (1.0 mol/L) was added and mixed with shaking to terminate the reaction, and the reaction product was cooled to room temperature and was used for sample injection to measure the formation of hippuric acid. The control tube was replaced with 10. Mu.L of 0.1mol/L boric acid-borax buffer (pH 8.3, containing 0.3mol/L NaCl) and the blank tube was then enzyme inactivated with 80. Mu.L HCl solution (1.0 mol/L) before the addition of the ACE solution. The reaction mixture was filtered through a 0.45 μm filter and analyzed by HPLC. High performance liquid chromatography conditions: welch Ultimate LC-C18 (HS) (250 mm. Times.4.6 mm,5 μm) column; the elution procedure was acetonitrile: ultrapure water=25:75 (containing 0.1% (v/v) TFA); flow rate: 1.0mL/min; detection wavelength: 228nm; column temperature: 30 ℃; sample injection amount: 20. Mu.L. The calculation formula is as follows:
Wherein: a is the chromatographic peak area of hippuric acid in the control group; b is the chromatographic peak area of hippuric acid in the sample group; a 0 is the chromatographic peak area of hippuric acid in the blank tube.
Example 1 obtaining selenium-enriched peptide for lowering blood pressure
1. Preparation of selenium-enriched oyster protein zymolyte
The selenium-enriched oyster protein hydrolysate is obtained by enzymolysis in the embodiment, and the specific process is as follows:
Dissolving selenium-enriched oyster protein powder into deionized water according to the mass concentration of the substrate of 5g/100mL, and regulating the pH value of the solution to 8.0. Trypsin was then added at an enzyme bottom ratio of 0.3%. After 3 hours of enzymolysis at the optimum temperature of 37 ℃, the enzymolysis solution is heated at 95 ℃ for 10 minutes to terminate the reaction. Cooling to room temperature, centrifuging at 4000r/min for 20min, collecting supernatant to obtain selenium-enriched oyster proteolytic liquid, and preserving at-20deg.C for use.
2. Purification and enrichment of antihypertensive selenium-rich peptide
(1) Ultrafiltration
Based on molecular weight, the selenium-enriched oyster protein hydrolysate was separated using an ultrafiltration tube (Amicon Ultra-15, molecular weight of filtration membrane 10 kDa), and centrifuged at 4000r/min for 30min. Fractions with molecular weights less than 10kDa were collected and stored at-20℃for subsequent analysis.
(2) Preparation of liquid chromatography
The ultrafiltration fraction (< 10 kDa) was subjected to preparative liquid chromatography using a Shimadzu PRC-ODS (K) steel column (30 mm. Times.250 mm,15 μm). The preparation conditions are as follows: mobile phase (A: primary water+0.1% TFA; B: methanol+0.1% TFA); elution procedure: 0-45 min, 5-10% of mobile phase B; 45-65 min, 10-20% of mobile phase B; 65-75 min, 20-50% of mobile phase B; 75-85 min, 95-95% of mobile phase B; 85-90 min, 95-5% of mobile phase B; sample injection amount is 5mL; the elution flow rate is 10mL/min; the detection wavelengths were 214nm and 280nm. The same elution peaks were pooled, concentrated by evaporation and lyophilized.
As shown in FIG. 1A, after purification of the ultrafiltered fraction by the preparative liquid phase, 5 main peaks were separated and designated as M1 to M5 fractions. The next step of separation and analysis is performed on M4.
The liquid phase system was prepared using Prep 150, and the reverse phase column SunFire Prep C18 OBD TM (19 mm. Times.250 mm,5 μm, waters) was used to further purify the most antioxidant component M4. The preparation conditions are as follows: mobile phase (A: double distilled water+0.1% TFA; B: methanol+0.1% TFA); gradient elution: 0-15 min, 6-8% of mobile phase B; 15-20 min, 8-40% of mobile phase B; 20-40 min, 40-50% of mobile phase B; 40-50 min, 95-95% of mobile phase B; 50-55 min, 95-6% of mobile phase B; sample injection amount is 1mL; the elution peak was monitored at an elution flow rate of 10mL/min at an ultraviolet wavelength of 214 nm. The active ingredient was collected, concentrated by rotary evaporation and lyophilized.
The secondarily prepared chromatogram is shown in FIG. 1B, the M4 fraction is further divided into 3 peaks (M4-1 to M4-3), and the fractions are collected and concentrated and freeze-dried for storage, wherein the M4-2 fraction is used for the subsequent analysis.
The selenium content of the active component in the purification and enrichment process of the antihypertensive selenium-rich peptide is measured, and the test results are shown in table 1. The test procedure was as follows: the selenium content of the sample is determined by referring to GB 5009.93-2017 method of atomic fluorescence spectrometry for determining hydride of selenium in food. The sample digestion adopts wet digestion, a proper amount of sample is weighed and placed in an conical flask, 10mL of nitric acid-perchloric acid mixed acid (v/v, 9:1) and a few glass beads are added, and the mixture is covered on a surface dish for cold digestion overnight. Heating on an electric heating plate the next day, and adding nitric acid in time. When the solution becomes clear and colorless with the generation of white smoke, the solution is continuously heated until the residual volume is about 2mL and can not be evaporated to dryness. Cooled, 5mL of hydrochloric acid solution (6.0 mol/L) was added, and heating was continued until the solution became clear and colorless with the appearance of white smoke. After cooling, the mixture was transferred to a 10mL volumetric flask, 2.5mL of a potassium iron oxide solution (100 g/L) was added, the volume was determined with water, and the mixture was mixed well to be measured. And simultaneously performing a reagent blank test.
The instrument conditions are as follows: negative high pressure 280V; the current of the selenium lamp is 80mA; the carrier gas flow rate is 300mL/min; the shielding gas flow is 800mL/min; reading time is 12s; delay time 3s; standard curve method of measuring mode.
At the same time, IC 50 values of ACE inhibitory activity were measured, and the test results are shown in Table 1.
TABLE 1 analysis of selenium content and IC 50 value for ACE inhibitory Activity
As is clear from Table 1, after trypsin enzymatic hydrolysis, the selenium content of the obtained enzymatic hydrolysate increased significantly to 2.44.+ -. 0.71mg/kg. After twice separation and purification, the selenium content of the component M4-2 with the highest ACE inhibition activity is up to 37.00+/-0.56 mg/kg, and is increased by 15.4 times compared with the selenium content of the zymolyte, which indicates that the selenium is well enriched in the separation and purification process.
(3) Identification of antihypertensive selenium-rich peptides
The desalted selenium-enriched peptide component with blood pressure reduction is centrifugally dried and then redissolved in deionized water containing 0.1% formic acid for online LC/MS analysis. The liquid phase was an Easy nLC 1200 nanoliter liquid phase system (ThermoFisher, USA), and the sample was run on a nanoViper C18 pre-column (3 μm,) Desalting, and purifying with C18 reversed phase chromatographic column (ACCLAIM PEPMAP RSLC,75 μm×25cm C18-2 μm /)) The gradient used for the separation was a 5% increase to 38% increase in mobile phase B (80% acetonitrile, 0.1% formic acid) over 60 min. The mass spectrum uses ThermoFisher Q Exactive system (ThermoFisher, USA) combined with Nano-liter spray Nano Flex ion source (ThermoFisher, USA), the spray voltage is 1.9kV, and the heating temperature of the ion transmission tube is 275 ℃. The mass spectrum scanning mode is in an information-dependent acquisition working mode (DDA, data Dependent Analysis), the primary mass spectrum scanning resolution is 70000, the scanning range is set to be 100-1500m/z, AGC TARGET is set to be 3 multiplied by 106, and the maximum injection time is 100ms. At most 20 secondary spectra with charges of 1+ to 3+ are acquired under each DDA cycle, the secondary mass spectrum AGC TARGET is set to 8000, and the maximum injection time is 50ms. High energy collision induced dissociation (HCD) was set to 28eV and dynamic exclusion was set to 6s. Data processing and search analysis were performed using de novo software PEAKSStudio 8.5.5 (Bioinformatics Solutions inc. Waters, canada). Searching Crassostrea gigas database, the local error discovery rate of PSM is 1.0%, and two cleavages are maximally missing. The search parameters were set to ：Oxidation(M),Acetylation(Protein N-term),Deamidation(NQ),Pyro-glu from E,Pyro-glu from Q,Carbamidomethylation(C) and Selenium replaces sulphur (MC) for variable modification. The precursor and fragment mass tolerance were 10ppm and 0.05Da, respectively.
The LC-MS/MS is adopted to analyze the polypeptide composition and the amino acid sequence of the active component M4-2, 91 selenium peptide sequences of ALC (%) more than or equal to 60 are totally identified, and the peptide sequences are all composed of 4-13 amino acid residues.
(4) Molecular docking screening antihypertensive selenium-rich peptide and interaction of antihypertensive selenium-rich peptide and ACE
The embodiment screens potential antihypertensive selenium-rich peptides based on a molecular docking technology. The process is as follows: the crystal structure of the receptor molecule 1O8A protein was downloaded from RCSB Protein Data Bank (www.rcsb.org). The water molecules and original ligands contained in the receptor molecules are removed by PyMol software, and Zn 2+ in ACE is added with positive charge of 0.95e, and the result is saved as a PDB format file. And then opening the PyMol treated acceptor molecule by using Autodock Tools 1.5.6 software, carrying out hydrotreatment, and storing the acceptor molecule as a pdbqt format file for later use. The two-dimensional structure of the ligand small molecule is drawn by MARVINSKETCH software, energy minimization structure optimization is carried out, the three-dimensional structure is output as a mol2 format file, the mol2 format file is opened by using Autodock Tools software, the ligand small molecule is hydrotreated, a Gasteiger is calculated, a rotary key is set, and the file is saved as pdbqt file. Grid Box center position (43.821, 38.24, 46.712), box size (100.125 × 100.125 × 100.125), exhaustiveness =20. Molecular docking was performed using Autodock Vina.1.2, and other parameters were default values unless specifically indicated. The result of the docking is expressed as the binding energy value, and the conformation with the smallest binding energy is selected as the optimal binding site. Finally, visual analysis of molecular docking results was performed using Discovery studio 4.5 software.
The results show that QASEMNEATGGK (1053.3994 Da) of the 91 peptides have low binding affinity to ACE, namely-9.0 kcal/mol, and are presumed to have potential ACE inhibitory activity. The selenium element QASEMNEATGGK exists in the form of SeMet (the sulfur element on methionine M in the peptide is replaced by selenium element), and the results of structure identification and molecular docking screening are shown in table 2.
TABLE 2 structural identification and molecular docking screening of selenium-enriched peptides for lowering blood pressure
To further explore the active mode of action of antihypertensive selenium-rich peptide QASEMNEATGGK, this example explores the binding mode and site of action between QASEMNEATGGK and the receptor ACE using molecular docking technology. Studies report that hydrogen bonding and hydrophobic interactions are two major interactions affecting ACE and inhibiting drug complex stability and inhibiting effect, and in addition Zn 2+ can coordinate with amino acid residues His383, his387 and Glu411 of ACE to form active center sites. Figure 2A shows the optimal docking conformation of QASEMNEATGGK to ACE, which the peptide is able to form a stable complex with ACE. Fig. 2B is a 2D schematic of the interaction of QASEMNEATGGK with amino acid residues of ACE. As shown in table 3, QASEMNEATGGK forms complexes with ACE primarily through hydrogen bonding, hydrophobic interactions, and metal-acceptor interactions. Molecular docking shows that the docking of QASEMNEATGGK to ACE forms hydrogen bonds at several sites Trp59, try62, asn66, asn70, leu139, ala356, his410, glu411, pro515, wherein binding to the site of Glu411 may to some extent distort tetrahedrally coordinated Zn 2+, thereby causing ACE to lose catalytic activity. Meanwhile, QASEMNEATGGK can also directly generate metal-acceptor action with the active center Zn701 of ACE to further block the combination of ACE and Zn 2+, thereby inhibiting the catalytic activity of ACE. Therefore, the binding of QASEMNEATGGK to the Glu411, zn701 sites of ACE results in the inhibition of ACE binding to Zn 2+, which may be critical for its ACE inhibitory activity. Furthermore, seMet of QASEMNEATGGK forms a hydrophobic interaction with amino acid residues Val518, pro519 of ACE, and the results indicate that the presence of elemental selenium may have a promoting effect on the binding of antihypertensive selenium-rich peptides to ACE, but the specific role of elemental selenium therein is to be studied intensively later.
TABLE 3 interaction and active site of antihypertensive selenium-rich peptides with ACE
(5) Synthesis of antihypertensive selenium-rich peptide
The screened antihypertensive selenium-rich peptide (QASEMNEATGGK) is synthesized by Nanje peptide biotechnology limited company, and has purity of more than 98% by High Performance Liquid Chromatography (HPLC) analysis, and is stored at-20deg.C.
Example 2 cell culture and cell viability assay of antihypertensive selenium-enriched peptides
(1) Detection of HepG2 cytotoxicity (MTT) by antihypertensive selenium-rich peptide
The culture of the ea.hy926 cells was referred to the existing method and was modified slightly. Ea.hy926 cells were inoculated into DMEM medium supplemented with 20% fetal bovine serum and 1% penicillin-streptomycin and incubated in an incubator containing 5% co 2 at 37 ℃. 100 mu L of EA.hy926 cell suspension (1X 10 5 cells/mL) is inoculated in a 96-well plate, the culture solution is discarded after the culture is carried out by adherence for 24 hours, 100 mu L of antihypertensive selenium-rich peptide solution (0.001, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25 and 0.5 mg/mL) is added into a sample group, a blank control group is replaced by a culture medium, and positive control group is captopril (0.5 mg/mL), and the culture is continued for 24 hours. After the packet treatment, cell viability was determined using the MTT method described by Mosmann. After the culture medium was discarded and washed with PBS, 100. Mu.L of MTT solution (0.5 mg/mL) was added thereto and incubation was continued for 4 hours. The MTT solution was discarded, 100. Mu.L of DMSO was added, and after shaking for 10min to dissolve the entire bluish violet crystals, the OD at 490nm was measured by an ELISA reader.
As shown in FIG. 3, when the concentration is less than or equal to 0.5mg/mL, the activity of QASEMNEATGGK treated cells is more than 90%, and the cells have no obvious toxic effect on EA.hy926 cells. Therefore QASEMNEATGGK can be used as safe ACE inhibitor within the concentration range of 0.001-0.5mg/mL, and has no cytotoxicity to vascular endothelial cells.
(2) Influence of antihypertensive selenium-rich peptide on NO secretion by cells
The ea.hy926 cells are a classical cell evaluation model commonly used in hypotensive activity studies. NO is an important mediator for regulating endothelial cell function, has a diastolic effect on vascular smooth muscle, and plays a very important role in balancing human blood pressure and maintaining vascular tension constant. Ea.hy926 cells were treated with different concentrations of antihypertensive selenium-rich peptide, captopril as a positive control, the results are shown in figure 4. Compared with the blank control group, the NO content of the blood pressure lowering selenium-enriched peptide treatment group gradually increases along with the increase of the treatment concentration, and when the peptide concentration reaches 0.025mg/mL, the NO content of the QASEMNEATGGK treatment group is increased by 273.45 percent and is obviously higher than that of the captopril positive control group (p < 0.05). Therefore, the antihypertensive selenium-rich peptide can promote EA.hy926 cells to generate NO, thereby playing an antihypertensive role and having a dosage effect.
(3) Influence of antihypertensive selenium-rich peptide on cell secretion of ET-1
In addition to the renin angiotensin system, the role of the endothelin system in blood pressure regulation is increasingly recognized, where ET-1 has powerful vasoconstrictor and boost properties. In the research, EA.hy926 cells are treated by the antihypertensive selenium-rich peptide, and the antihypertensive activity of the antihypertensive selenium-rich peptide can be reflected by detecting the change of the ET-1 level. As can be seen from fig. 5, the antihypertensive selenium-rich peptide group had a significant inhibitory effect on the production of cell ET-1 (p < 0.05) compared with the blank group (109.72 ±1.66 pg/mL) after 24h treatment, and there was a significant dose dependency between the low, medium and high dose groups. The content of ET-1 in the high dose group (0.025 mg/mL) treatment group is (79.15 +/-2.32 pg/mL) which is significantly lower than that in the captopril positive control group (88.67+/-0.91 pg/mL) (p < 0.05). The experimental result shows that the antihypertensive selenium-rich peptide has good antihypertensive effect on the cellular level.
In conclusion, QASEMNEATGGK has NO obvious toxic effect on EA.hy926 cells at a certain concentration, and can promote the generation of NO and inhibit the secretion of ET-1. The blood pressure lowering selenium-rich peptide is shown to exert the blood pressure lowering effect by increasing the release amount of NO and reducing the secretion of ET-1.
Finally, it should be noted that the above embodiments are merely representative examples of the present invention. Obviously, the technical solution of the invention is not limited to the above-described embodiments, but many variations are possible. All modifications which can be derived or suggested directly from the present disclosure by a person skilled in the art should be considered as the protection scope of the claims of the present invention.
Claims (8)
1. The antihypertensive selenium-rich peptide is characterized by being SEQ ID NO:1, and the sulfur element in methionine M in the antihypertensive selenium-rich peptide is replaced by selenium.
2. The use of the antihypertensive selenium-rich peptide of claim 1 in the preparation of antihypertensive drugs.
3. The use according to claim 2, wherein the blood pressure lowering drug is a drug that promotes the release of NO.
4. The use according to claim 2, wherein the blood pressure lowering drug is a drug that inhibits the secretion of ET-1.
5. The use according to claim 2, wherein the blood pressure lowering drug is an ACE inhibitor.
6. The use according to claim 5, wherein the antihypertensive selenium-rich peptide is used for the preparation of an ACE inhibitor which binds to amino acid residues Trp59, try62, asn66, asn70, leu139, ala356, his410, glu411, pro515 of ACE by hydrogen bonding.
7. The use according to claim 5, wherein the antihypertensive selenium-rich peptide is used for the preparation of ACE inhibitors that bind to amino acid residues Val518, pro519 of ACE via hydrophobic interactions.
8. The use according to claim 5, wherein the antihypertensive selenium-rich peptide is used for preparing an ACE inhibitor which inhibits the binding of ACE to Zn 2+.
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