CN114195857A - Antihypertensive peptide, and preparation method and application thereof - Google Patents
Antihypertensive peptide, and preparation method and application thereof Download PDFInfo
- Publication number
- CN114195857A CN114195857A CN202111200166.4A CN202111200166A CN114195857A CN 114195857 A CN114195857 A CN 114195857A CN 202111200166 A CN202111200166 A CN 202111200166A CN 114195857 A CN114195857 A CN 114195857A
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- ace
- peptide
- spirulina
- antihypertensive peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
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- A—HUMAN NECESSITIES
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- A61K38/00—Medicinal preparations containing peptides
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Abstract
The invention belongs to the technical field of biology, and particularly relates to antihypertensive peptide and application thereof in medicines and functional food health-care foods. The invention takes spirulina as raw material, uses proteinase K to carry out enzymolysis to spirulina protein, and the oligopeptide with the sequence of TVLYEH is obtained by the separation of the polypeptide after the enzymolysis through gel chromatography and reversed phase chromatography. Research shows that the polypeptide has obvious Angiotensin Converting Enzyme (ACE) inhibiting activity and IC502.59. mu.M, and no significant cytotoxicity. The invention provides a method for preparing antihypertensive peptide, successfully identifies high-activity oligopeptide, and has good development and utilization prospects.
Description
Technical Field
The invention belongs to the technical field of bioactive peptides, and relates to application of a polypeptide in the research and development of an Angiotensin Converting Enzyme (ACE) inhibitor or foods, medicines or health-care products related to hypertension treatment.
Background
Bioactive peptides (Bioactive peptides) are peptide fragments with certain bioactivity consisting of 2-20 amino acid units. The polypeptide with the activities of resisting tumor, resisting bacteria, resisting inflammation, reducing blood sugar, resisting virus, reducing blood pressure and the like is separated from soybean, gluten, casein and aquatic product protein. By 9 months 2021, the BIOPEP database contains up to 4300 or more polypeptides with various biological activities, 1046 polypeptides with ACE inhibitory activity, and the number of the polypeptides is the largest. Compared with macromolecular protein, the bioactive peptide has the characteristics of small molecular weight, easy absorption, low antigenicity and the like, and is widely applied to the fields of food, health-care products, cosmetics, medicines and the like.
Hypertension refers to Systolic Blood Pressure (SBP) >140mmHg and/or Diastolic Blood Pressure (DBP) >90mmHg without the use of a hypotensive agent, and is classified into 1 grade, 2 grades, and 3 grades according to the level of elevation of blood pressure. As a chronic multiple disease, the health and daily life of a large number of patients are disturbed, researches prove that the blood pressure level and the cardiovascular disease risk are in direct relation, and long-term hypertension can also damage target organs such as heart, large blood vessel, kidney, eye, brain and the like. The prevalence rate of hypertension of adults in China is 23.2 percent and still shows an ascending trend, so that effective hypertension treatment medicines and health-care foods have very high research values.
In the regulation of blood pressure, the Renin-Angiotensin System (RAS) and the Kinin-Bradykinin System (KKS) play a crucial role. In the RAS system, angiotensinogen is hydrolyzed by renin to produce angiotensin I, and further, after hydrolysis catalyzed by Angiotensin Converting Enzyme (ACE), angiotensin II is produced, which acts on the corresponding receptor to cause vasoconstriction and thus blood pressure rise. In addition, ACE may also catalyze the degradation of bradykinin and decrease the secretion of NO and prostaglandins through a cascade of reactions, thereby mitigating the effects of nitric oxide and prostaglandin-induced blood pressure elevation. In conclusion, ACE plays a crucial role in the regulation of blood pressure, and the regulation of its activity is crucial for the control of blood pressure. The development of ACE inhibitors has therefore played a major role in the prevention and treatment of hypertension.
The existing blood pressure regulating medicines comprise diuretics, prils, terraces and sartans. These antihypertensive drugs, while regulating blood pressure, reduce the patient's body compliance with the drugs and thus increase the cost of treatment and the difficulty of blood pressure control. There is therefore a need to find more diverse ACE inhibitors. In the past decades, natural polypeptides with the activities of reducing blood pressure and blood sugar, resisting bacteria, regulating immunity and the like are separated from zymolytes of milk, soybeans and fish proteins. The naturally derived ACE inhibitory peptides have higher safety compared with chemical synthesis drugs while effectively generating a regulating effect on the activity of ACE, and although some ACE inhibitory peptides derived from spirulina are reported at present, polypeptides with better activity and higher safety are still to be discovered.
The spirulina has a long history of utilization as a nutrient-rich alga, is rich in nutrients and contains various nutrient substances such as protein, vitamins, trace elements necessary for human bodies and the like. Research shows that the spirulina has the functions of resisting fatigue, lowering blood pressure, resisting bacteria, resisting virus, resisting oxidation and the like. The spirulina protein is possible to release small molecule active peptide with better activity and easier absorption through enzymolysis. Some patents related to spirulina active peptides exist at present, and most of the patents are preparation methods of the active peptides, such as patents with publication numbers of CN111154824A, CN107502641A, CN107674905A, CN103981245A, CN107446977A, CN101906135A, CN10126546 and CN 112646856A. The activity of the polypeptides reported in these patents is focused on the aspects of antioxidation, antibiosis, fatigue resistance and the like, and except that the antihypertensive peptide with the sequence of IQP is reported in the patent with the publication number of CN101906135A by Lujun, the antihypertensive peptide with clear spirulina is not needed. Therefore, the spirulina antihypertensive peptide still needs to be further developed and researched.
Disclosure of Invention
The invention aims to provide antihypertensive peptide and application thereof in food, medicines or health-care products.
In order to achieve the purpose, the invention adopts the technical scheme that:
the antihypertensive peptide is obtained by carrying out enzymolysis and further separation on Spirulina Spirulina platensis or Spirulina maxima serving as raw materials, has a sequence of Thr-Val-Leu-Tyr-Glu-His (TVLYEH), and has a molecular weight of 760.35 Da. The polypeptide obtained by an enzymolysis and separation method or a chemical synthesis method can be applied to the preparation of antihypertensive drugs, pharmaceutical compositions or health products.
The antihypertensive peptide can obviously inhibit Angiotensin Converting Enzyme (ACE) under the in-vitro experiment condition that the equoyl-histidine-leucine is used as a substrate, and the half inhibition rate (IC50) is 2.59 mu M.
The preparation method of the antihypertensive peptide comprises the following steps: the ratio of material to water is 1: 10-1: 20, after the suspension is frozen at the temperature of-20 ℃, the suspension is unfrozen at the temperature of 20-50 ℃, the feed liquid is treated by using an ultrasonic cell disruption instrument with the power of 200-800w, after the suspension is circulated for three times, the suspension is centrifuged, the supernatant is taken, and protease K is added according to the ratio of 1-10% of enzyme to substrate, and the enzymolysis conditions are as follows: performing enzymolysis at 25-65 deg.C for 1-12 hr at 8-12 deg.C, and heating at 95 deg.C for 15min to inactivate enzyme. And (3) using a Sephadex G-15 gel column, using deionized water as a mobile phase, collecting an elution peak by using an automatic fraction collector at the flow rate of 1mL/min, and freeze-drying to obtain the spirulina antihypertensive peptide.
The polypeptide can be combined with an active pocket of angiotensin converting enzyme, and inhibition kinetic data show that the polypeptide is a competitive inhibitor of ACE.
The medicine, health-care product or food prepared by the conventional preparation method can realize the treatment, alleviation or prevention of hypertension after the polypeptide is taken as an active ingredient and is mixed with auxiliary materials meeting the production requirements of medicines or foods.
The invention has the advantages that:
the polypeptide obtained by the invention has excellent in-vitro ACE inhibitory activity and has no obvious cytotoxicity in a cell level experiment. The polypeptide is hexapeptide, has molecular weight of 760.35Da, small molecular weight, easy absorption, and certain gastrointestinal digestive enzyme stability. In conclusion, the polypeptide has good application prospect in the fields of foods, health-care products, medicines and the like with blood pressure regulating activity, and researches show that the polypeptide has remarkable angiotensin-converting enzyme (ACE) inhibitory activity and IC502.59. mu.M, and no significant cytotoxicity.
Drawings
Figure 1 gel chromatography separates the component ACE inhibitory activity.
Figure 2 reverse phase chromatography separates the component ACE inhibitory activity.
FIG. 3 is a 2D diagram of TVLYEH-ACE interaction
FIG. 4 is a TVLYEH mass spectrum.
FIG. 5TVLYEH purity determination liquid chromatogram.
FIG. 6 ACE inhibition of TVLYEH at different concentrations.
FIG. 7 is a double reciprocal plot of TVLYEH.
FIG. 8 the effect of TVLYEH on the proliferative capacity of mouse macrophage RAW 264.7.
Detailed Description
The invention is further explained below with reference to the figures and examples. The present invention is directed to the use of spirulina as a starting material, which is proteolytically processed, isolated and screened for a defined sequence, and it is understood that these examples are intended to be illustrative of the present invention and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the disclosure of the present invention, and such equivalents also fall within the scope of the invention.
Example 1
High Performance Liquid Chromatography (HPLC) method for determining in vitro ACE inhibitory activity of polypeptide
The experimental method comprises the following steps:
the principle of the method is as follows: the hippuroyl-histaminoyl-leucine (Hip-His-Leu, HHL, Sigma-Aldrich) can be used as a substrate of Angiotensin Converting Enzyme (ACE) to be decomposed to generate hippuric acid, after different ACE inhibitors are added, the generation amount of hippuric acid is correspondingly reduced, and the inhibitory activity of the inhibitor on the ACE activity can be evaluated by calculating the peak area of hippuric acid at 228 nm.
Experimental reagent:
ACE (0.1U/mL), hippuryl-histidine-leucine (HHL), captopril, 0.1M sodium borate solution (pH8.3, containing 0.3M sodium chloride)
ACE was dissolved in sodium borate buffer to a final concentration of 0.1U/mL for assay. Polypeptide samples are dissolved in sodium borate buffer solution to prepare polypeptide solutions with different concentrations. Then, 20. mu.L of a polypeptide solution of a certain concentration was mixed with 10. mu.L of an ACE solution. The mixture was incubated at 37 ℃ for 5min, and 50. mu.L of 5mM HHL (sodium borate buffer pH8.3, containing 0.3M sodium chloride) was added to the mixture to start the reaction. The reaction was maintained at 37 ℃ for 60min and then 150. mu.L of 1M HCl molar was added to stop the reaction. The solution was passed through a 0.22 μm filter to obtain a reaction solution. mu.L of the reaction solution was loaded into RP-HPLC, which was connected to an Eclipse XDB-C18 column (4.6 mm. times.150 mm. times.5 μm), and the concentration of Hippuric Acid (HA) was measured by UV. Hippuric acid absorbance was measured at 228 nm. All absorbance measurements were performed in triplicate. ACE inhibitory activity was calculated as follows:
ACE inhibitory activity (%) - (AControl-AInhibitor)/AControl 100)
Wherein AInhibitor is the relative area of Hippuric Acid (HA) peaks from the reaction of ACE and HHL with inhibitors. AControl is the relative area of the Hippuric Acid (HA) peak obtained from the reaction of ACE and HHL without inhibitor. IC50 is defined as the concentration of polypeptide that inhibits half of the ACE activity.
Chromatographic conditions are as follows:
c18 column (4.6mm x 150mm x 5 μm, Agilent), detection wavelength: 228 nm; mobile phase: 78% ultrapure water (containing 0.05% TFA) + 22% acetonitrile (containing 0.05% TFA); flow rate: 0.8 mL/min.
Example 2
Preparation of spirulina antihypertensive peptide
Weighing 15g of spirulina dry powder to disperse in 240mL of deionized water, freezing for 4h at-20 ℃, unfreezing at 37 ℃, and then crushing spirulina suspension by using an ultrasonic cell crusher, wherein the parameters of the ultrasonic cell crusher are set to work for 15s at an interval of 15s, and the power is 550W for ultrasonic treatment for 60 min. The temperature of the container is controlled at 0-4 ℃ during ultrasonic treatment, and the container is placed on ice to prevent the liquid from being heated up due to ultrasonic treatment. Performing freeze thawing-ultrasonic circulation for 3 times, then 10000RCF, centrifuging at 4 ℃ for 10min, and taking the supernatant.
Measuring protein concentration, adjusting the protein concentration to 10mg/mL by adding deionized water, adjusting the pH to 10 by adding 0.1M NaOH solution, adding proteinase K according to 4.5% of the protein mass content, reacting at 57 ℃ for 4h, heating at 95 ℃ for 15min for inactivating enzyme, centrifuging at 12000g for 10min, taking supernatant, and freeze-drying to obtain the crude spirulina antihypertensive peptide extract. According to the determination, the ACE inhibition rate of the spirulina antihypertensive peptide is 76.44% when the concentration is 400 mu g/mL.
Gel chromatography separation purification and activity evaluation of (II) spirulina antihypertensive peptide
The crude extract of the spirulina antihypertensive peptide is separated and purified by a gel chromatography column (Sephadex G15, 1.6 multiplied by 100cm), deionized water is used as a mobile phase, the flow rate is controlled to be 1mL/min, and four components with the elution time of 80-86min, 87-112min, 113-138min and 139-162min are collected, freeze-dried and named as SK-G1, SK-G2, SK-G3 and SK-G4 respectively.
Evaluation of ACE inhibitory activity of 4 components was performed according to the calculation method of ACE inhibitory activity in example 1, and the ACE inhibitory rate of SK-G4 was 86.85% at a concentration of 400. mu.g/mL.
(III) reverse chromatographic separation and purification and activity evaluation of spirulina antihypertensive peptide
SK-G4 SK-G4 separated from Sephadex G-15 was separated on an Agilent Zorbax SB-Aq C18 column (4.6X 250mm, 5 μm) by the following elution procedure:
1-5 min: 5% acetonitrile (v%); 5-55 min: 5% -95% acetonitrile (v%); 55-60 min: 95% acetonitrile (v%); the flow rate was 0.8 mL/min. 12 fractions were collected according to the following table for peak time.
TABLE 1 elution time component comparison Table
| SK-G4R1 | SK-G4R2 | SK-G4R3 | SK-G4R4 | SK-G4R5 | SK-G4R6 |
| 4-5min | 6-7min | 8-9min | 10-11min | 12-15min | 16-17min |
| SK-G4R7 | SK-G4R8 | SK-G4R9 | SK-G4R10 | SK-G4R11 | SK-G4R12 |
| 17-18min | 19-20min | 21-22min | 23-25min | 32-33min | 59-60min |
The ACE inhibitory activity of 12 components was evaluated according to the method of example 1, and three components, SK-G4R2, SK-G4R3, and SK-G4R5, had relatively good ACE inhibitory activity.
(IV) identification of spirulina antihypertensive peptide sequence
Dissolving a sample in a proper amount of ddH2O, adding DTT to enable the final concentration to be 10mmol/L, adding an IAA solution to enable the final concentration to be 50mmol/L after water bath at 56 ℃ for 1h, carrying out light-shielding reaction for 40min, desalting, volatilizing the solvent in vacuum, and redissolving by using a 0.1% formic acid solution for LC-MS/MS analysis.
Nano LC-MS/MS: the packing material of the chromatographic column is Repuril-Pur C18-AQ (1.9 μm,
) The specification was 150. mu. m.times.150 mm. The mobile phase A, B was water containing 0.1% formic acid and acetonitrile, respectively, and was subjected to gradient elution at a flow rate of 600nL/min after loading 5. mu.L. Gradient program as follows:
TABLE 2 elution gradiometer
| Time (min) | |
| 0 | 4％ |
| 2 | 8％ |
| 45 | 28％ |
| 55 | 40％ |
| 56 | 95％ |
| 66 | 95％ |
A plurality of polypeptide sequences including the polypeptide with the sequence TVLYEH are obtained by aligning secondary mass spectrum data obtained by Q active Hybrid Quadrupole-Orbitrap-MS/MS (Thermo Fisher Scirnitic, USA) in a Byonic software self-contained database.
Example 3
Virtual screening of spirulina antihypertensive peptides
The resulting polypeptide sequences were subjected to a first round of screening based on abundance. Two-and three-dimensional structures for each polypeptide were mapped using ChemDraw. The polypeptide structure is subjected to protonation and energy minimization at pH7.0, and then is stored and subjected to molecular docking with a human tACE crystal structure (PDB ID: 1O8A) by using Pyrx, wherein the active center zinc ion is taken as the center of a docking box during docking, and the radius of a docking sphere is
The docking results show the affinity of each polypeptide with ACE, wherein the docking score of TVLYEH is-9.0, and the affinity is stronger. TVLYEH has 93.08% inhibition of ACE at a mass concentration of 200. mu.g/mL as measured by the method described in example 1.
As can be seen from the interaction 2D plot (fig. 3), TVLYEH and Asn66, Asn70, Gln281, Thr282 and His353 within the ACE active site form hydrogen bonds, occupy the ACE active center, and can compete with the substrate for the ACE active site.
Example 4
Mass spectrometric identification of TVLYEH
0.1mg of the sample is dissolved in 0.5mL of ultrapure water, the sample passes through a C18 column and is analyzed by a Q-active mass spectrometer, the sample loading amount is 1 mu L, the carrier gas flow rate is 1.5L/min, and the liquid phase mobile phase is 50% H2O + 50% MeOH.
The molecular weight of TVLYEH is 760.35Da, as in mass spectrum (FIG. 4).
Example 5
Purity identification of TVLYEH
0.5mg of the sample was dissolved in 0.5mL of ultrapure water and analyzed by a high performance liquid chromatography system equipped with NanoChrom Chromcore TM 120C 18(4.6 MM 250MM 5. mu.M) chromatography column. The loading was 40. mu.L, and the mobile phases were acetonitrile containing 0.1% trifluoroacetic acid and ultrapure water, respectively. The flow rate was 1.0mL/min, and a peak was detected at 214 nm.
The purity of TVLYEH is not less than 95% by analyzing the polypeptide chromatographic peak by normalization method, and the chromatographic peak is shown in FIG. 5.
Example 6
IC50 determination of TVLYEH
The ACE inhibition of TVLYEH was measured as described in example 1 at concentrations of 100, 50, 10, 1, 0.1, 0.01, 0.001 μ g/mL, three replicates were set up and the mean was plotted as log concentration versus inhibition to calculate the IC50 value for the polypeptide.
TABLE 3 antihypertensive peptides and IC reported in the prior patents50
The IC50 value of the spirulina antihypertensive peptide TVLYEH is 2.59 mu M, and the activity is higher than that of the antihypertensive peptide reported in the prior patent. The curve of ACE inhibition as a function of polypeptide concentration is shown in FIG. 6.
Example 7
Inhibition patterns of TVLYEH
HHL solutions with concentrations of 4, 2, 1, 0.5, 0.25, 0.1mM and polypeptide solutions with concentrations of 0.5mg/mL and 0.1mg/mL were prepared with boric acid buffer, and the activity evaluation as described in example 1 was performed using the polypeptide with different concentrations and the HHL solutions with different concentrations, respectively. The inverse of the rate of hippuric acid production was plotted against the inverse of substrate concentration, and the inhibition pattern of the polypeptide against ACE was analyzed by the double reciprocal method.
As shown in fig. 7, as the concentration of the polypeptide increases, the slope of the reciprocal curve increases while the vertical intercept is unchanged, i.e., Vmax is unchanged, Km is increased, and the curve intersects the vertical axis, so that the inhibition pattern of the polypeptide is considered to be competitive inhibition, which is also consistent with the effect of TVLYEH on the ACE activity center in the virtual screening.
Example 8
Toxicity evaluation of TVLYEH
Experimental methods
Blowing and beating RAW264.7 cells in a logarithmic growth phase into a single-cell suspension, after cell counting, inoculating the RAW264.7 cells into a 96-well plate according to the cell density of 5 multiplied by 105/mL for culturing, and when the cells grow to about 50%, replacing an old culture medium with 100 mu L of blank or a culture medium containing TVLYEH with different concentrations, and continuing culturing for 24 hours. mu.L of 5mg/mL MTT solution was added to each well, incubation was continued for 4h, the supernatant was discarded, 150. mu.L DMSO was added to each well, the crystals were dissolved by gentle shaking at room temperature for 10min, and the absorbance was measured at 490 nm.
Cell viability was ═ (experimental OD value/blank OD value) x 100%.
The results are shown in FIG. 8, when TVLYEH was incubated alone on RAW264.7 cells, there was no significant inhibition of cell proliferation at concentrations of 50-400. mu.g/mL.
Sequence listing
<110> oceanographic institute of Chinese academy of sciences
<120> antihypertensive peptide, preparation method and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Thr Val Leu Tyr Glu His
1 5
Claims (6)
1. An antihypertensive peptide characterized by: the polypeptide has an amino acid sequence shown in a sequence table SEQ ID NO. 1; specifically, the sequence is Thr-Val-Leu-Tyr-Glu-His (abbreviated as TVLYEH).
2. Use of the antihypertensive peptide according to claim 1, wherein: the polypeptide is spirulina polypeptide, and the spirulina polypeptide has an inhibitory effect on Angiotensin Converting Enzyme (ACE) as an active ingredient.
3. A method for preparing the antihypertensive peptide according to claim 1, which comprises: the feed-liquid ratio (g: mL) of the spirulina dry powder to water is 1: 10-1: 20, after suspension is frozen for 2-8h at the temperature of-20 ℃, the suspension is unfrozen and is treated by using an ultrasonic cell disruption instrument, after circulation (freeze thawing and ultrasonic cell disruption) is carried out for three times, the suspension is centrifuged, supernatant is taken, protease K is added according to the mass ratio of 1-10% of enzyme substrate, and the enzymolysis conditions are as follows: carrying out enzymolysis at 25-65 deg.C for 1-12h at pH8-12, heating at 95 deg.C for 15min to inactivate enzyme to obtain zymolyte, and separating the zymolyte by gel column chromatography and reversed phase column chromatography to obtain the antihypertensive peptide.
4. Use of a hypotensive peptide, wherein: the antihypertensive peptide is applied to the preparation of Angiotensin Converting Enzyme (ACE) inhibitors or foods, health-care products or pharmaceutical preparations related to the prevention of hypertension diseases, the alleviation of hypertension diseases or the treatment of hypertension diseases.
5. An angiotensin converting enzyme inhibitor or a preparation for the treatment of hypertension, the alleviation of hypertension or the prevention of hypertension, characterized in that: the antihypertensive peptide according to claim 1 as an active ingredient.
6. The formulation of claim 5, wherein: the peptide or peptide extract of claim 1, in combination with any carrier or excipient acceptable in food or pharmaceutical manufacture.
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| CN115197309A (en) * | 2022-07-15 | 2022-10-18 | 广东南兴天虹果仁制品有限公司 | Amygdalus communis protein-derived ACE inhibitory peptide, and preparation method and application thereof |
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| EP1914550A1 (en) * | 2001-05-07 | 2008-04-23 | HiPep Laboratories | Peptide-immobilized substrate and method for measuring target protein using the same |
| CN108840909A (en) * | 2018-07-24 | 2018-11-20 | 中国科学院海洋研究所 | A kind of laver antihypertensive peptide and laver antihypertensive peptide extract and application |
| CN108892710A (en) * | 2018-07-24 | 2018-11-27 | 中国科学院海洋研究所 | Asparagus is depressured peptide extract and asparagus Antihypertensive Peptides and its application |
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| EP1914550A1 (en) * | 2001-05-07 | 2008-04-23 | HiPep Laboratories | Peptide-immobilized substrate and method for measuring target protein using the same |
| CN108840909A (en) * | 2018-07-24 | 2018-11-20 | 中国科学院海洋研究所 | A kind of laver antihypertensive peptide and laver antihypertensive peptide extract and application |
| CN108892710A (en) * | 2018-07-24 | 2018-11-27 | 中国科学院海洋研究所 | Asparagus is depressured peptide extract and asparagus Antihypertensive Peptides and its application |
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| CN115197309A (en) * | 2022-07-15 | 2022-10-18 | 广东南兴天虹果仁制品有限公司 | Amygdalus communis protein-derived ACE inhibitory peptide, and preparation method and application thereof |
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