CN113943346B - Antihypertensive peptide of spirulina and application - Google Patents
Antihypertensive peptide of spirulina and application Download PDFInfo
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- CN113943346B CN113943346B CN202111200168.3A CN202111200168A CN113943346B CN 113943346 B CN113943346 B CN 113943346B CN 202111200168 A CN202111200168 A CN 202111200168A CN 113943346 B CN113943346 B CN 113943346B
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- spirulina
- antihypertensive
- peptide
- ace
- polypeptide
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- Peptides Or Proteins (AREA)
Abstract
The invention belongs to the technical field of biology, and relates to a preparation method of spirulina antihypertensive active peptide and application of spirulina antihypertensive active peptide in functional foods, health products or medicines. The invention uses spirulina as raw material, uses proteinase K to make enzymolysis to spirulina protein, and makes the polypeptide after enzymolysis undergo the processes of gel chromatography and reversed phase chromatography separation so as to obtain spirulina oligopeptide with sequences of TVFNHEGR and LQAGGLF. Experiments show that the isolated polypeptide has remarkable Angiotensin Converting Enzyme (ACE) inhibitory activity, and IC50 is 198 mu M and 60.01 mu M respectively. The invention provides a method for preparing spirulina antihypertensive peptide, and successfully identifies two spirulina oligopeptides with good antihypertensive activity and novel sequence, provides a reference for fully utilizing spirulina protein resources, 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 spirulina polypeptides in research and development of Angiotensin Converting Enzyme (ACE) inhibitors or foods, medicines or health care products related to hypertension treatment.
Background
The bioactive peptide (Bioactive peptides) is a peptide segment with certain bioactivity and consists 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 obtained by separating from soybean, gluten, casein and aquatic product proteins. By 9 months 2021, the bipep database contains up to 4300 more polypeptides with various biological activities, of which the polypeptides with ACE inhibitory activity are 1046, the number being the largest. Compared with macromolecular proteins, the bioactive peptide has the characteristics of small molecular weight, easy absorption, low antigenicity and the like, and based on the bioactive peptide, the bioactive peptide is widely applied to the fields of foods, health care products, cosmetics, medicaments and the like.
Hypertension refers to Systolic Blood Pressure (SBP) >140mmHg and/or Diastolic Blood Pressure (DBP) >90mmHg without hypotensor and is classified into class 1, class 2, and class 3 according to the level of elevation of blood pressure. As a chronic multiple disease afflicts the physical health and daily life of a wide range of patients, studies have demonstrated that blood pressure levels and cardiovascular disease risk present a direct relationship. In addition, long-term hypertension can cause damage to target organs such as heart, large blood vessels, kidneys, eyes, brain, and the like. The prevalence of hypertension of adults in China is 23.2 percent and still shows rising trend, so that the effective antihypertensive drugs and health-care foods have very research value.
During blood pressure regulation, the Renin-angiotensin system (Renin-Angiotensin System, RAS) and the Kinin-bradykinin system (kinen-Bradykinin System, KKS) play a vital role. In the RAS system, angiotensinogen is hydrolyzed by renin to produce angiotensin I, and further, angiotensin II is produced by catalytic hydrolysis of Angiotensin Converting Enzyme (ACE), which acts on the corresponding receptor to cause vasoconstriction and thus blood pressure rise. In addition, ACE can also catalyze the degradation of bradykinin and reduce the secretion of NO and prostaglandins through a series of cascade reactions, thereby alleviating the effects of elevated blood pressure caused by nitric oxide and prostaglandins. In summary, ACE plays a vital role in the blood pressure regulation process, and regulation of its activity is vital to the control of blood pressure. Thus, development of ACE inhibitors plays a major role in the prevention and treatment of hypertension.
The existing blood pressure regulating drugs are favorable for diuretics, polycosanolides, terraces and sartans. The antihypertensive drugs can reduce the compliance of a patient body to the drugs while regulating the blood pressure, thereby increasing the treatment cost and the difficulty of blood pressure control. There is therefore a need to find a greater variety of ACE inhibitors. Over the past decades, natural polypeptides with antihypertensive, hypoglycemic, antibacterial, immunoregulatory activities have been isolated from the enzymatic hydrolysates of milk, soy and fish proteins. While the ACE inhibitory peptides of natural sources effectively regulate the activity of ACE, the ACE inhibitory peptides have higher safety than chemical synthetic drugs, some of ACE inhibitory peptides derived from spirulina are reported at present, but polypeptides with better activity and higher safety are yet to be discovered.
The spirulina has very long utilization history as a nutrient-rich alga, is rich in nutrients, and is rich in various nutrients such as proteins, vitamins, trace elements necessary for human bodies and the like. The research shows that the spirulina has the functions of resisting fatigue, reducing blood pressure, resisting bacteria, resisting viruses, resisting oxidation and the like. The spirulina protein can release small molecular active peptide with better activity and easier absorption after enzymolysis. There are some patents related to spirulina active peptide, and most of the patents are the preparation methods of active peptide, such as patent publication numbers CN111154824A, CN107502641A, CN107674905A, CN103981245A, CN107446977A, CN101906135A, CN10126546, CN112646856a, etc. The activities of the polypeptides reported in these patents are focused on antioxidant, antibacterial, antifatigue aspects, and the like, and besides the spirulina antihypertensive peptide with the sequence of IQP reported in the patent with the publication number of CN101906135A, the spirulina antihypertensive peptide with clear unordered sequence is reported in Lu Jun. Thus, spirulina antihypertensive peptides still remain to be further developed and studied.
In terms of antihypertensive peptides, published patents and polypeptide sequences with exact sequence information are shown in Table 1, and both polypeptides required by the patent are different from the polypeptides.
Table 1 shows the published patents and polypeptide sequences for antihypertensive peptides.
Disclosure of Invention
The invention aims to provide spirulina antihypertensive peptide and application thereof in foods, medicines or health care products.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the antihypertensive peptide is obtained by taking Spirulina Spirulina platensis or spirorina maxima as a raw material through enzymolysis and further separation, and the Spirulina polypeptide has an amino acid sequence shown in a sequence table SEQ ID NO. 1; the specific sequence is Thr-Val-Phe-Asn-His-Glu-Gly-Arg (abbreviated as TVFNHEGR).
The spirulina polypeptide is an amino acid sequence in a sequence table SEQ ID NO. 2; the specific sequence is Leu-Gln-Ala-Gly-Gly-Leu-Phe ((abbreviated as LQAGGLF).
Wherein the molecular weight of the sequence of the polypeptide is Thr-Val-Phe-Asn-His-Glu-Gly-Arg (TVFNHEGR) and Leu-Gln-Ala-Gly-Gly-Leu-Phe (LQAGGLF) is 959.03Da and 704.82Da respectively. The polypeptide obtained by enzymolysis, separation or chemical synthesis can be applied to the preparation of a antihypertensive drug, a pharmaceutical composition or a health product.
The spirulina antihypertensive peptide can obviously inhibit Angiotensin Converting Enzyme (ACE) under the in vitro experimental condition of taking the maleyl-histidine-leucine as a substrate, and the half inhibition rate (IC 50) is 190.3 mu M and 60.01 mu M.
The preparation method of the spirulina antihypertensive peptide comprises the following steps: the ratio of the spirulina dry powder to the water feed liquid (g: mL) is 1:10-1:20, the suspension is frozen at the temperature of minus 20 ℃ and thawed at the temperature of 20-50 ℃, the feed liquid is processed by an ultrasonic cell disruption instrument with the power of 200-800w, the supernatant is centrifugally taken after three times of circulation (freeze thawing and ultrasonic cell disruption), proteinase K is added into the supernatant according to the enzyme bottom ratio of 1% -10%, and the enzymolysis conditions are as follows: and (3) carrying out enzymolysis at 25-65 ℃ for 1-12h at pH 8-12, and then heating at 95 ℃ for 15min to inactivate enzymes. And (3) using a Sephadex G-15 gel column, using deionized water as a mobile phase, using an automatic fraction collector to collect an elution peak, and freeze-drying to obtain the spirulina antihypertensive peptide.
The spirulina polypeptide is taken as an active ingredient, and after being mixed with auxiliary materials meeting the production requirements of medicines or foods, the medicines, health products or foods obtained by the conventional preparation method can realize the treatment, alleviation or prevention effects on hypertension.
The spirulina polypeptide is one or two spirulina antihypertensive peptides or spirulina antihypertensive peptide extracts in the sequence table SEQ ID NO.1 or SEQ ID NO.2, and is prepared by adding any carrier or auxiliary materials which meet the production allowance of foods or medicines.
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, the molecular weight is 959.03Da and 704.82Da respectively, and the polypeptide is small in molecular weight and easy to absorb. The polypeptide has good application prospect in the fields of foods, health care products, medicaments and the like with blood pressure regulating activity.
Drawings
FIG. 1TVFNHEGR, LQAGGLF mass spectrum.
FIG. 2 is a purity-identifying liquid chromatogram of FIG. TVFNHEGR, LQAGGLF.
Fig. 3 ACE inhibition rates for different concentrations TVFNHEGR, LQAGGLF.
Fig. 4LQAGGLF double reciprocal curve.
The patent and polypeptide sequences of antihypertensive peptides disclosed in Table 1.
Detailed Description
The invention is further illustrated by the following figures and examples. The purpose of the present invention is to take spirulina as raw material, through proteolytic hydrolysis, separation and screening with a definite sequence, it should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications of the invention may be made by those skilled in the art after reading the disclosure of the invention, and such equivalents are intended to fall within the scope of the invention as defined by the claims.
Example 1
Determination of in vitro ACE inhibitory Activity of Polypeptides by High Performance Liquid Chromatography (HPLC)
The experimental method comprises the following steps:
the principle of the method is as follows: the hippocampal-histamine-leucine (Hip-His-Leu, HHI, 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 the hippuric acid is correspondingly reduced, and the inhibition activity of the inhibitor on the ACE activity can be evaluated by calculating the peak area of the hippuric acid at 228 nm.
Experimental reagent:
ACE (0.1U/mL), maleyl-histidine-leucine (HHI), captopril, 0.1M sodium borate solution (pH 8.3, 0.3M sodium chloride)
ACE was dissolved in sodium borate buffer to a final concentration of 0.1U/mL for the assay. Polypeptide samples are dissolved in sodium borate buffer 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 ACE solution. The mixture was incubated at 37℃for 5min, and 50. Mu.L of HHT (sodium borate buffer pH8.3, containing 0.3M sodium chloride) at a molar concentration of 5mM was added to the mixture to initiate the reaction. The reaction was maintained at 37℃for 60min, and then 150. Mu.L of HCl having a molar concentration of 1M was added to terminate 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 on RP-HPLC, which was coupled to Eclipse XDB-C18 column (4.6 mm. Times.150 mm. Times.5 μm), and the concentration of Hippuric Acid (HA) was detected by ultraviolet. Hippuric acid was detected as absorbance at 228 nm. All absorbance measurements were repeated three times. ACE inhibition activity was calculated as follows:
ACE inhibitory activity (%) = (AControl-AInhibitor)/AControl 100
Wherein AInhibitor is the relative area of the peak of Hippuric Acid (HA) obtained by reaction of ACE and HIL with the inhibitor. AControl is the relative area of the Hippuric Acid (HA) peak obtained from the reaction of ACE and HHL without inhibitors. IC50 is defined as the concentration of polypeptide that can inhibit half of ACE activity.
Chromatographic conditions:
c18 column (4.6mm x 150mm x 5 μm, agilent), detection wavelength: 228nm; mobile phase: 78% ultrapure water (containing 0.05% TFA) +22% acetonitrile (containing 0.05% TFA); flow rate: 0.8mL/min.
Example 2
Preparation of spirulina antihypertensive peptide
15g of spirulina dry powder is weighed to be dispersed in 240mL of deionized water, frozen for 4h at minus 20 ℃, thawed at 37 ℃, and the spirulina suspension is crushed by an ultrasonic cytoclasis instrument, wherein the parameters of the ultrasonic cytoclasis instrument are set to work for 15s, the interval is 15s, and the power is 550W and the ultrasonic is 60min. The container is placed on ice to control the temperature at 0-4 ℃ during ultrasonic treatment, so that the liquid is prevented from being heated up due to ultrasonic treatment. Freezing thawing-ultrasonic circulation is carried out for 3 times, 10000RCF is carried out, and centrifugation is carried out for 10min at 4 ℃ to obtain supernatant.
Measuring the protein concentration, regulating the protein concentration to 10mg/mL by adding deionized water, regulating the pH to 10 by adding a NaOH solution with the concentration of 0.1M, adding proteinase K according to 4.5% of the protein mass content, reacting for 4 hours at 57 ℃, heating for 15min at 95 ℃, inactivating enzyme, centrifuging for 10min by 12000g, and freeze-drying the supernatant to obtain the spirulina antihypertensive peptide crude extract. The measurement shows that the ACE inhibition rate of the spirulina antihypertensive peptide is 76.44% at the concentration of 400 mug/mL.
Gel chromatography separation and purification and activity evaluation of spirulina antihypertensive peptide
Separating and purifying crude extract of spirulina antihypertensive peptide with gel chromatographic column (Sephadex G15, 1.6X100 cm), taking deionized water as mobile phase, controlling flow rate at 1mL/min, collecting and freeze-drying four components with elution time of 80-86min,87-112min,113-138min and 139-162min, named SK-G1, SK-G2, SK-G3 and SK-G4 respectively.
The ACE inhibitory activity of 4 components was evaluated according to the ACE inhibitory activity calculation method in example 1, with SK-G4 having an ACE inhibition rate of 86.85% at a concentration of 400 μg/mL.
Reverse chromatographic separation and purification and activity evaluation of spirulina antihypertensive peptide
SK-G4 the SK-G4 separated from Sephadex G-15 was separated on an Agilent Zorbax SB-Aq C18 column (4.6X250 mm,5 μm) with the following elution procedure:
1-5min:5% acetonitrile (v%); 5-55min:5% -95% acetonitrile (v%); 55-60min:95% acetonitrile (v%); the flow rate was 0.8mL/min. The 12 fractions were collected corresponding to the peak times as shown in the following table.
TABLE 2 elution time composition 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 the 12 components was evaluated according to the method in example 1, and three components SK-G4R2, SK-G4R3, SK-G4R5 had relatively good ACE inhibitory activity.
Identification of spirulina antihypertensive peptide sequence
The sample was dissolved in an appropriate amount of ddH2O and DTT was added to a final concentration of 10mmol/L, IAA solution was added to a final concentration of 50mmol/L after 1h of water bath at 56℃and the reaction was carried out for 40min in the dark, desalted, and redissolved with 0.1% formic acid solution after vacuum evaporation of the solvent for LC-MS/MS analysis.
Nano LC-MS/MS: the column packing was ReproSil-Pur C18-AQ (1.9 μm,
) The specification is 150 μm by 150mm. Mobile phases A, B were water and acetonitrile containing 0.1% formic acid, respectively, and 5 μl was loaded and then subjected to gradient elution at a flow rate of 600 nL/min. Gradient procedure is as follows:
TABLE 3 elution gradient Table
| Time (min) | |
| 0 | 4％ |
| 2 | 8％ |
| 45 | 28％ |
| 55 | 40％ |
| 56 | 95％ |
| 66 | 95％ |
Secondary mass spectrometry data obtained using Q Exactive Hybrid Quadrupole-Orbitrap-MS/MS (Thermo Fisher Scirntific, USA) were aligned in a Byonic software self-contained database to obtain multiple polypeptide sequences including polypeptides with sequences tvfnhlegr and LQAGGLF.
Example 3
Virtual screening of spirulina antihypertensive peptides
And carrying out first-round screening on the obtained polypeptide sequence according to the abundance. The two and three dimensional structure of each polypeptide was mapped using ChemDraw. The polypeptide structure is preserved and subjected to molecular docking with human tACE crystal structure (PDB ID:1O 8A) by using Pyrx after protonation and energy minimization at pH7.0, and when docking, active center zinc ion is used as the center of a docking box, and the radius of a docking sphere is equal to
The docking results showed affinity of each polypeptide to ACE, with a docking score of-9.0 for TVFNHEGR, LQAGGLF, with a stronger affinity. TVFNHEGR, LQAGGLF exhibits 68.4% and 64.09% inhibition of ACE, respectively, at a concentration of 400 μg/mL as measured in example 1.
Example 4
Mass spectrometry identification of TVFNHEGR, LQAGGLF
0.1mg of the sample was dissolved in 0.5mL of ultrapure water, and after passing through a C18 column, the sample was analyzed by a Q-exact mass spectrometer with a loading of 1. Mu.L, a carrier gas flow rate of 1.5L/min, and a liquid mobile phase of 50% H2O+50% MeOH.
As shown in FIG. 1, mass spectrometry identified two polypeptides TVFNHEGR, LQAGGLF with molecular weights of 959.03Da and 704.82Da, respectively, in sequence.
Example 5
Purity identification of TVFNHEGR, LQAGGLF
0.5mg of the sample was weighed and dissolved in 0.5mL of ultrapure water, and then the sample was analyzed by a high performance liquid chromatography system equipped with a NanoChrom Chromcore TM C18 (4.6X1250 MM. Times.5. Mu.M) column. The loading was 40. Mu.L, and the mobile phases were acetonitrile and ultrapure water containing 0.1% trifluoroacetic acid, respectively. The flow rate was 1.0mL/min, and the peak was detected at 214nm, and the chromatogram was shown in FIG. 2.
The polypeptide chromatographic peaks are analyzed by a normalization method, and the purity of the TVFNHEGR and LQAGGLF polypeptides is more than or equal to 95 percent.
Example 6
IC50 determination of TVFNHEGR, LQAGGLF
The ACE inhibition of TVFNHEGR, LQAGGLF was determined as described in example 2 at a concentration of 100, 50, 10,1,0.1,0.01,0.001 μg/mL, three replicates were set up, and the IC50 value of the polypeptide was calculated by plotting the average value as the log of the concentration versus the inhibition (fig. 3).
The IC50 values of spirulina antihypertensive peptide TVFNHEGR, LQAGGLF were measured to be 190.3. Mu.M and 60.01. Mu.M, respectively.
Example 7
Inhibition pattern of LQAGGLF A solution of HHT at 4,2,1,0.5,0.25,0.1mM and a solution of polypeptide at 0.5mg/mL and 0.1mg/mL were prepared in boric acid buffer, and the activity evaluation as described in example 2 was performed by taking different concentrations of polypeptide and different concentrations of HHT solution, respectively. The inhibition pattern of ACE by the polypeptides was analyzed by the double reciprocal method, plotted as the reciprocal of the rate of hippuric acid production versus the reciprocal of the substrate concentration.
As shown in fig. 4, the slope of the double reciprocal curve increases with increasing concentration of the polypeptide and the vertical intercept becomes constant, i.e., vmax becomes constant Km, and the curves intersect on the vertical axis, so that the inhibition pattern of the polypeptide is considered to be competitive inhibition, which also coincides with the result of the action of LQAGGLF with ACE active center in virtual screening.
Sequence listing
<110> national academy of sciences of China sea institute
<120> spirulina antihypertensive peptide and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Thr Val Phe Asn His Glu Gly Arg
1 5
<210> 2
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Leu Gln Ala Gly Gly Leu Phe
1 5
Claims (5)
1. A spirulina antihypertensive peptide, which is characterized in that: the amino acid sequence of the spirulina antihypertensive peptide is shown in a sequence table SEQ ID NO. 1.
2. A spirulina antihypertensive peptide, which is characterized in that: the amino acid sequence of the spirulina antihypertensive peptide is shown in a sequence table SEQ ID NO. 2.
3. Use of the spirulina antihypertensive peptide according to claim 1 or 2, characterized in that: use of one or both of the spirulina antihypertensive peptides of claim 1 or claim 2 as an active ingredient in the preparation of an Angiotensin Converting Enzyme (ACE) inhibitor or in the preparation of a pharmaceutical formulation for the prevention of hypertension, the alleviation of hypertension or the treatment of hypertension.
4. An agent for the treatment of an angiotensin converting enzyme inhibitor or a hypertension, the alleviation of a hypertension or the prevention of a hypertension, characterized in that: one or two of the spirulina antihypertensive peptides as claimed in claim 1 or claim 2 are used as active ingredients.
5. The formulation of claim 4, wherein: is prepared by one or two spirulina antihypertensive peptides or extracts containing the spirulina antihypertensive peptides according to claim 1 or claim 2 and any carrier or auxiliary materials which meet the production allowance of foods or medicines.
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