CN114195857B

CN114195857B - Antihypertensive peptide and preparation method and application thereof

Info

Publication number: CN114195857B
Application number: CN202111200166.4A
Authority: CN
Inventors: 张全斌; 索齐善; 岳洋; 王晶; 吴宁; 耿丽华
Original assignee: Institute of Oceanology of CAS
Current assignee: Institute of Oceanology of CAS
Priority date: 2021-10-14
Filing date: 2021-10-14
Publication date: 2023-07-04
Anticipated expiration: 2041-10-14
Also published as: CN114195857A

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 uses spirulina as raw material, uses proteinase K to make enzymolysis to spirulina protein, and uses gel chromatography and reversed phase chromatography to separate the polypeptide after enzymolysisAn oligopeptide with a sequence of TVLLYEH was obtained. Research shows that the polypeptide has obvious Angiotensin Converting Enzyme (ACE) inhibiting activity and IC ₅₀ 2.59. Mu.M, with no significant cytotoxicity. The invention provides a method for preparing antihypertensive peptide, and successfully identifies a high-activity oligopeptide, thereby having good development and utilization prospects.

Description

Antihypertensive peptide and preparation method and application thereof

Technical Field

The invention belongs to the technical field of bioactive peptides, and relates to application of a polypeptide in developing an Angiotensin Converting Enzyme (ACE) inhibitor or food, medicine or health care product 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, the health and daily life of a wide patient are plagued, researches prove that the blood pressure level and the cardiovascular disease risk are in direct relation, and in addition, long-term hypertension can damage target organs such as heart, great 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 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.

Disclosure of Invention

The invention aims to provide 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 and performing enzymolysis and further separation, and has a sequence of Thr-Val-Leu-Tyr-Glu-His (TVLLYEH) and a molecular weight of 760.35Da. 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 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 2.59 mu M.

The preparation method of the antihypertensive peptide comprises the following steps: the ratio of the material to the water is 1:10-1:20, the suspension is frozen at the temperature of minus 20 ℃ and thawed at the temperature of 20-50 ℃ and treated by an ultrasonic cell disruption instrument with the power of 200-800w, the supernatant is centrifugally taken after three times of circulation, proteinase K is added in the supernatant at 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 pH8-12, 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 polypeptides are capable of binding to the active pocket of angiotensin converting enzyme, and inhibition kinetics data indicate that the polypeptides are competitive inhibitors of ACE.

The 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 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 a molecular weight of 760.35Da, has small molecular weight, is easy to absorb, and has certain gastrointestinal tract 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 IC ₅₀ 2.59. Mu.M, with no significant cytotoxicity.

Drawings

Figure 1 gel chromatography separates the ACE inhibitory activity of components.

Figure 2 reverse phase chromatography separates the ACE inhibitory activity of the components.

FIG. 3 TVLLYEH-ACE interaction 2D diagram

Fig. 4TVLYEH mass spectrum.

FIG. 5 TVLLYEH purity characterization liquid chromatogram.

Fig. 6 ACE inhibition rates at different concentrations of TVLYEH.

Fig. 7TVLYEH double reciprocal curve.

FIG. 8 effect of TVLLYEH on proliferation potency of mouse macrophage RAW 264.7.

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 temperature of the container is controlled to be 0-4 ℃ during ultrasonic treatment, and the container is placed on ice to prevent the liquid from rising temperature 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 1 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 2 elution gradient table

Time (min)	Phase B
		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 of sequence TVLYEH.

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 TVLYEH, with a strong affinity. TVLLYEH has 93.08% inhibition of ACE as measured by the method described in example 1 at a mass concentration of 200. Mu.g/mL.

From the interaction 2D plot (FIG. 3), it can be seen that TVLLYEH and Asn66, asn70, gln281, thr282 and His353 within the ACE active site form hydrogen bonds, occupy the active center of ACE, and can compete with the substrate for the active site of ACE.

Example 4

Mass spectrometry identification of TVLLYEH

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.

For example, the mass spectrum (FIG. 4) shows that TVLLYEH has a molecular weight of 760.35Da.

Example 5

Purity identification of TVLLYEH

0.5mg of the sample was weighed and dissolved in 0.5mL of ultrapure water, and then analyzed by a high performance liquid chromatography system equipped with a NanoChrom Chromcore TM C18 (4.6X 250MM X5. 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 peak conditions were detected at 214 nm.

The polypeptide chromatographic peak is analyzed by a normalization method, the purity of TVLLYEH is more than or equal to 95%, and the chromatographic peak is shown in figure 5.

Example 6

IC50 determination of TVLLYEH

The ACE inhibition of TVLYEH was determined as described in example 1 at a concentration of 100, 50, 10,1,0.1,0.01,0.001 μg/mL, and the IC50 value of the polypeptide was calculated by plotting the average value as the log of the concentration versus the inhibition in triplicate.

TABLE 3 antihypertensive peptides and ICs reported in the prior patents ₅₀

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 ACE inhibition rate as a function of polypeptide concentration is shown in FIG. 6.

Example 7

Inhibition pattern of TVLLYEH

A4, 2,1,0.5,0.25,0.1mM HHT solution and a 0.5mg/mL and 0.1mg/mL polypeptide solution were prepared in boric acid buffer, and the activities of the polypeptides at different concentrations and the HHT solutions at different concentrations were evaluated as described in example 1. 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. 7, as the concentration of the polypeptide increases, the slope of the double reciprocal curve becomes larger and the vertical intercept becomes unchanged, that is, vmax becomes unchanged 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 effect result of TVLYEH on the ACE active center in the virtual screening.

Example 8

Toxicity evaluation of TVLLYEH

Experimental method

The RAW264.7 cells in the logarithmic phase are blown into single cell suspension, after cell counting, the RAW264.7 cells are inoculated into a 96-well plate for culture according to the cell density of 5 multiplied by 105/mL, when the cells grow to about 50%, 100 mu L of blank or culture medium containing TVLyEH with different concentrations is used for replacing the old culture medium, and the culture is continued for 24 hours. To each well, 20. Mu.L of MTT solution at a concentration of 5mg/mL was added, incubation was continued for 4 hours, the supernatant was discarded, 150. Mu.L of DMSO was added to each well, and the crystals were sufficiently dissolved by gentle shaking at room temperature for 10 minutes, and absorbance was measured at a wavelength of 490 nm.

Cell viability= (experimental OD value/placebo OD value) ×100%.

As a result, as shown in FIG. 8, TVLyEH showed no significant inhibition of cell proliferation at concentrations of 50-400. Mu.g/mL when incubated with RAW264.7 cells alone.

Sequence listing

<110> national academy of sciences of China sea institute

<120> antihypertensive peptide, and 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

1. A antihypertensive peptide, characterized in that: the antihypertensive peptide is an amino acid sequence shown in a sequence table SEQ ID NO. 1; the sequence is Thr-Val-Leu-Tyr-Glu-His (abbreviated as TVLLYEH).

2. A process for the preparation of the antihypertensive peptide according to claim 1, which comprises: the ratio g of the spirulina dry powder to the water is 1:10-1:20, after freezing the suspension for 2-8 hours at the temperature of minus 20 ℃, thawing, treating the feed liquid by using an ultrasonic cell disruption instrument, and centrifuging to obtain the supernatant after three times of circulation of freeze thawing and ultrasonic cell disruption, adding proteinase K in the supernatant according to the mass ratio of 1% -10% of enzyme bottom, wherein the enzymolysis conditions are as follows: and (3) carrying out enzymolysis at 25-65 ℃ for 1-12h at pH8-12, heating at 95 ℃ for 15min, inactivating enzyme to obtain an enzymolysis product, and separating the enzymolysis product by gel column chromatography and reverse phase column chromatography to obtain the antihypertensive peptide.

3. Use of a antihypertensive peptide of claim 1 for the preparation of an Angiotensin Converting Enzyme (ACE) inhibitor or for the preparation of a pharmaceutical formulation for the prevention of hypertension or for the alleviation of hypertension or for 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: the antihypertensive peptide of claim 1 as an active ingredient.

5. The formulation of claim 4, wherein: is prepared by the antihypertensive peptide of claim 1 and any carrier or auxiliary materials which are allowed by food or medicine production.