CN110028550B - Blood pressure lowering peptide and blood pressure lowering protein and their application - Google Patents

Abstract

The invention discloses a blood pressure lowering peptide, a blood pressure lowering protein and an application thereof; the amino acid sequence of the blood pressure lowering peptide is KAKW, and the amino acid sequence of the blood pressure lowering protein contains KAKW; the blood pressure lowering peptide and the blood pressure lowering protein can inhibit angiotensin The activity of invertase can lower blood pressure, and can be used to make various blood pressure lowering drugs or health care products.

Description

Blood pressure lowering peptide and blood pressure lowering protein and their application

technical field

The invention belongs to the technical field of food or medicine, and relates to a blood pressure-lowering peptide and a blood pressure-lowering protein and applications thereof.

Background technique

Angiotensin converting enzyme (ACE) is an enzyme that can increase blood pressure, and it has the following two regulatory pathways.

First, angiotensin-converting enzyme can excise two amino acids (His-Leu) at the end of angiotensin I (Angiotensin I), thereby converting the angiotensin I into angiotensin II (Angiotensin II). Among them, the amino acid sequence of angiotensin I is Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu, abbreviated as DRVYIHPFHL. The amino acid sequence of angiotensin II is Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, abbreviated as DRVYIHPF. Angiotensin II is a vasoconstrictor that promotes the constriction of blood vessels, thereby increasing blood pressure.

Second, angiotensin-converting enzyme can cleave two amino acids (Phe-Arg) at the end of bradykinin (Bradykinin), thereby inactivating it. Wherein, the amino acid sequence of bradykinin is Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, and the amino acid sequence of inactivated bradykinin is Arg-Pro-Pro-Gly-Phe-Ser-Pro . Bradykinin has the function of dilating blood vessels, but when bradykinin is inactive, it loses the ability to dilate blood vessels, which increases blood pressure.

ACE is a metallopeptidase that contains a Zn ²⁺ binding site, which is the "obligatory binding site" for substrate binding. The Zn ²⁺ binding site is the site where the active group of ACE catalyzes the reaction. The common action of various ACE inhibitors is to bind to the Zn ²⁺ binding site of ACE and inactivate it.

If the angiotensin-converting enzyme is inactivated, the angiotensin-converting enzyme cannot convert angiotensin I to angiotensin II, and the blood vessels will not contract; at the same time, the angiotensin-converting enzyme cannot make bradykinin Inactivated, blood vessels dilate. The combined effect of the two will lower blood pressure. Therefore, finding a polypeptide or protein that can inactivate angiotensin-converting enzyme has become an urgent technical problem to be solved.

SUMMARY OF THE INVENTION

One of the objects of the present invention is to provide a blood pressure-lowering peptide capable of inhibiting angiotensin-converting enzyme.

Another object of the present invention is to provide the application of the above-mentioned blood pressure lowering peptide.

In order to achieve the above object, the solution of the present invention is:

A blood pressure lowering peptide whose amino acid sequence is TTW, is called the first blood pressure lowering peptide.

A blood pressure lowering peptide whose amino acid sequence is VHW is called the second blood pressure lowering peptide.

A blood pressure lowering peptide whose amino acid sequence is shown in SEQ ID NO: 1 is called the third blood pressure lowering peptide.

Any of the above-mentioned blood pressure-lowering peptides can be used as angiotensin-converting enzyme inhibitors.

Any of the above-mentioned blood pressure lowering peptides can be used to prepare blood pressure lowering drugs.

Any of the above-mentioned blood pressure lowering peptides can be used to prepare blood pressure lowering health care products.

A blood pressure-lowering protein containing one or more of TTW, VHW and the amino acid sequence shown in SEQ ID NO:1.

The above-mentioned blood pressure-lowering proteins can be used as angiotensin-converting enzyme inhibitors.

The above-mentioned hypotensive proteins can be used for preparing hypotensive drugs.

The above-mentioned blood pressure lowering protein can be used for preparing blood pressure lowering health care products.

Owing to adopting the above-mentioned scheme, the beneficial effects of the present invention are:

The blood pressure-lowering peptide of the present invention is obtained by screening from the hydrolyzate of natural food, and has obvious inhibitory activity on angiotensin-converting enzyme. Combined use of state-of-the-art antihypertensive drugs in order to achieve better synergistic antihypertensive effect.

Description of drawings

FIG. 1 is a schematic diagram of the hydrogen bond formation between the first blood pressure-lowering peptide of the present invention and the active center of angiotensin converting enzyme.

Figure 2 is a schematic diagram of the hydrogen bond formation between the second blood pressure-lowering peptide of the present invention and the active center of angiotensin converting enzyme.

3 is a schematic diagram of the hydrogen bond formation between the third blood pressure-lowering peptide of the present invention and the active center of angiotensin converting enzyme.

Figure 4 is a graph showing the antihypertensive effect of blood pressure-lowering peptides.

Detailed ways

The present invention provides multiple blood pressure lowering peptides and their applications.

<First blood pressure lowering peptide>

The present invention provides a blood pressure lowering peptide, the blood pressure lowering peptide is composed of three amino acids, and its amino acid sequence is Thr-Thr-Trp, abbreviated as TTW. For convenience of explanation, hereinafter collectively referred to as the first blood pressure-lowering peptide. The amino acids in the present invention are arranged from the N-terminus to the C-segment.

Molecular docking experiments showed that the first T in the first blood pressure-lowering peptide formed three hydrogen bonds with Ala354 in the active center of angiotensin-converting enzyme (ACE); the second T in the first blood pressure-lowering peptide was His353, Glu384 and His513 each form a hydrogen bond; W in the first blood pressure lowering peptide forms a hydrogen bond with Glu403, so the first blood pressure lowering peptide forms a total of 7 hydrogen bonds with angiotensin converting enzyme (ACE). The formation of these seven hydrogen bonds is shown in Figure 1. The amino acids in the circle represent the amino acid sequence in ACE, and the chain in the middle is the configuration formed by the sequence of the blood pressure-lowering peptide in ACE.

These amino acid residues are all important amino acid residues in the active center of angiotensin-converting enzyme (ACE), and have an important influence on the activity of ACE. When the first blood pressure-lowering peptide forms a hydrogen bond with these amino acid residues in the active center of angiotensin-converting enzyme (ACE), the active center of angiotensin-converting enzyme (ACE) can no longer bind angiotensin, thereby vascular Tensin-converting enzyme (ACE) is then inactivated, so this first blood pressure-lowering peptide can act as an angiotensin-converting enzyme inhibitor.

<Application of the first blood pressure lowering peptide>

1. The first blood pressure lowering peptide can be used to prepare blood pressure lowering drugs.

The first blood pressure lowering peptide of the present invention can be used alone as a blood pressure lowering drug, at this time, the first blood pressure lowering peptide needs to be used in combination with auxiliary materials to make drug granules, capsules, tablets (such as sugar-coated tablets or film-coated tablets), Pills, oral liquids or injections, etc.

When the first blood pressure-lowering peptide is used in pharmaceutical granules, the excipients can be maltodextrin and/or vanillin.

When the first blood pressure lowering peptide is used as a sugar-coated tablet, the excipients can be starch and/or magnesium stearate.

When the first blood pressure-lowering peptide is used as an oral liquid, the excipients can be sodium benzoate, aspartame, acesulfame potassium, essence and purified water.

When the first blood pressure-lowering peptide is used as an injection, the excipients can be mannitol, sodium dihydrogen phosphate and/or disodium hydrogen phosphate. The injection can be formulated as 0.9% sodium chloride injection or 5% dextrose injection followed by intravenous drip or intramuscular injection.

The first blood pressure lowering peptide of the present invention can also be used in combination with the blood pressure lowering drugs of the prior art, so as to obtain a better synergistic blood pressure lowering effect.

Antihypertensive drugs in the prior art include beta blockers, calcium antagonists, ACE inhibitors and diuretics.

Wherein, the beta-blocker is selected from nebivolol, pravolol, arolol, atenolol, celibetol, carvedilol, labetalol, and bisoprolol.

The calcium antagonist is selected from the group consisting of nifedipine, amlodipine (including levoamlodipine), lacadipine, felodipine, nilvadipine, lacidipine, nisoldipine.

The ACE inhibitor is selected from the group consisting of alapril, benazepril, captopril, cilopril, cilazapril, delapril, enalapril, enalapril, fosinopril, Lisinopril, Ramipril, Ramipril, Perindopril, Quinapril, Spiropril, Temopril, Trandopril.

The diuretic is selected from hydrochlorothiazide, trichlorothiazide.

2. The first blood pressure lowering peptide can be used to prepare blood pressure lowering health care products.

The first blood pressure lowering peptide of the present invention can be made into a blood pressure lowering health care product by adding auxiliary materials, and the auxiliary materials can include: starch, gelatin, titanium dioxide, whey protein, sucrose, vitamin E, glutathione, arginine, citron Amino acid, dietary fiber, collagen, cephalin, cellulose, glucose, xylose, honey, lecithin, carotenoid, vitamin B1, vitamin B2, vitamin C, iron, calcium, bee pollen, black fungus powder ( 200-500 mesh), mushroom powder (300-500 mesh), Ganoderma lucidum powder (300-500 mesh), konjac powder (300-500 mesh), astragalus powder (200-500 mesh), wolfberry powder (200-500 mesh) And any one or more of ginseng powder (500-1000 mesh), etc.

<Second blood pressure lowering peptide>

The present invention provides a blood pressure lowering peptide, the blood pressure lowering peptide is composed of three amino acids, and its amino acid sequence is Val-His-Trp, abbreviated as VHW. For convenience of explanation, hereinafter collectively referred to as the second blood pressure lowering peptide.

Molecular docking experiments showed that V in the second blood pressure-lowering peptide formed two hydrogen bonds with Glu411 in the active center of angiotensin-converting enzyme (ACE); A hydrogen bond is formed; W in the second blood pressure lowering peptide forms a hydrogen bond with Tyr520, so the second blood pressure lowering peptide and angiotensin converting enzyme (ACE) form a total of 7 hydrogen bonds. The formation of these seven hydrogen bonds is shown in Figure 2.

These amino acid residues are also important amino acid residues in the active center of angiotensin-converting enzyme (ACE), and have an important influence on the activity of ACE. When the second blood pressure-lowering peptide forms hydrogen bonds with these amino acid residues in the active center of angiotensin-converting enzyme (ACE), the active center of angiotensin-converting enzyme (ACE) can no longer bind angiotensin, thereby vascular The tensin-converting enzyme (ACE) is then inactivated, so this second blood pressure-lowering peptide can also act as an angiotensin-converting enzyme inhibitor.

<Application of the second blood pressure lowering peptide>

1. The second blood pressure lowering peptide can be used to prepare blood pressure lowering drugs.

2. The second blood pressure lowering peptide can be used to prepare blood pressure lowering health care products.

Wherein, when using the second blood pressure lowering peptide to prepare a blood pressure lowering drug or a blood pressure lowering health care product, the auxiliary materials that can be used in combination with the second blood pressure lowering peptide can refer to the first blood pressure lowering peptide.

<Third blood pressure lowering peptide>

The present invention provides a blood pressure lowering peptide, the amino acid sequence of which is shown in SEQ ID NO: 1. For the convenience of explanation, hereinafter collectively referred to as the third blood pressure lowering peptide. The third blood pressure lowering peptide consists of four amino acids, and its amino acid sequence is Lys-Ala-Lys-Trp, abbreviated as KAKW.

Molecular docking showed that the first K in the third antihypertensive peptide formed two hydrogen bonds with Ala356 in the active center of ACE and one hydrogen bond with Glu411; the W in the third antihypertensive peptide formed with Gln281 Two hydrogen bonds were formed, one hydrogen bond was formed with Tyr520 and Asp415; the second K in the third blood pressure-lowering peptide formed one hydrogen bond with His353, Lys511, so the third blood pressure-lowering peptide and angiotensin Invertase (ACE) forms a total of 9 hydrogen bonds. The formation of these nine hydrogen bonds is shown in Figure 3. These amino acid residues are all important amino acid residues in the active center of angiotensin-converting enzyme (ACE), and have an important influence on the activity of ACE. When the third blood pressure-lowering peptide forms hydrogen bonds with these amino acid residues in the active center of angiotensin-converting enzyme (ACE), the active center of angiotensin-converting enzyme (ACE) can no longer bind angiotensin, thereby vascular Tensin-converting enzyme (ACE) is then inactivated, so this third blood pressure-lowering peptide can act as an angiotensin-converting enzyme inhibitor.

<Application of the third blood pressure lowering peptide>

1. The third blood pressure lowering peptide can be used to prepare blood pressure lowering drugs.

2. The third blood pressure lowering peptide can be used to prepare blood pressure lowering health care products.

Wherein, when using the third blood pressure lowering peptide to prepare a blood pressure lowering drug or a blood pressure lowering health care product, the excipients that can be used in combination with the third blood pressure lowering peptide can refer to the first blood pressure lowering peptide.

<blood pressure lowering protein>

A blood pressure lowering protein is characterized in that: it contains one or more of TTW, VHW and the amino acid sequence shown in SEQ ID NO: 1.

The above-mentioned polypeptides and proteins can be synthesized by artificial methods, so the synthesis methods are not repeated here.

<Application of blood pressure lowering protein>

1. The antihypertensive protein can be used to prepare antihypertensive drugs.

2. The blood pressure lowering protein can be used to prepare blood pressure lowering health care products.

When using a blood pressure lowering protein to prepare a blood pressure lowering drug or a blood pressure lowering health care product, reference can be made to the first blood pressure lowering peptide for the excipients that can be used in combination with the blood pressure lowering protein.

The blood pressure-lowering activity of various blood pressure-lowering peptides and the like will be described below in conjunction with experiments.

<Experiment 1: In vitro inhibition experiment of various blood pressure-lowering peptides on angiotensin-converting enzyme>

The various blood pressure-lowering peptides of the present invention belong to competitive inhibitors with strong affinity to the active region of angiotensin converting enzyme, and their affinity to the active region is higher than that of angiotensin I or bradykinin, and once bound It is also not easily released from the binding area, which can reduce the activity of the active area of ACE, or even inactivate it, thereby preventing ACE from converting angiotensin I to angiotensin II, and hindering blood vessels. Tensin-converting enzyme inactivates bradykinin, which in turn acts to lower blood pressure. The inhibitory ability of various blood pressure-lowering peptides on angiotensin-converting enzyme was verified experimentally below.

The measurement principle of this experiment is as follows:

Hip-His-Leu (HHL for short) can serve as a substrate of angiotensin-converting enzyme (ACE) and be hydrolyzed into hippuric acid and His-Leu. Inhibitors can inhibit the ability of angiotensin-converting enzyme to catalyze the hydrolysis of hippuric acid histidyl leucine and reduce the content of hippuric acid in the product. Therefore, the inhibitor can be calculated by detecting the content of hippuric acid in the product. Angiotensin-converting enzyme inhibition rate.

The measurement steps of this experiment are as follows:

(1), take multiple EP tubes as reaction vessels, add 20 μL of deionized water to the EP tubes of the control group, and add 20 μL of samples to be tested with different concentrations to the EP tubes of the experimental group;

(2), add 80 μL of 5M hippuryl histidyl leucine (HHL) to each EP tube, and place in a water bath at 37° C. for 5 minutes;

(3), add 10 μL of 310mU/mL angiotensin-converting enzyme (ACE) to each EP tube, and start the reaction in a water bath at 37°C for 5 minutes;

(4), when the reaction is completed, add 400 μL 1 M of HCL to each EP tube to terminate the reaction;

(5), filter the reaction solution in each EP tube with a filter membrane of 0.22 μm respectively, and store the filtered liquid in the corresponding liquid phase vial;

(6), perform high performance liquid chromatography detection on the liquid in each liquid phase vial, and the detection conditions are: chromatographic column: Thermo BDSHYPERSIL C18 (250mm×3mm×5μm); column temperature: 30°C; flow rate: 0.8mL/min; Detection wavelength: 228 nm; injection volume: 10 μL; mobile phase A: ultrapure water (0.1% trifluoroacetic acid TFA); mobile phase B: acetonitrile; elution conditions are shown in Table 1.

Table 1 HPLC elution conditions

(7), the calculation method of the inhibition rate of each sample to be tested to angiotensin-converting enzyme is as follows:

X=(A control-A)/A control

In the formula: X——ACE inhibition rate (%);

A control - the peak area of the control group;

A - the peak area of the experimental group.

(8) Calculation of IC50 of the sample to be tested:

Taking the concentration of the sample to be tested as the abscissa and the ACE inhibition rate as the ordinate, plotting the inhibition rate-sample concentration relationship curve, the concentration of the sample to be tested when the inhibition rate reaches 50% is obtained from the curve, which is the test sample to be tested. IC50 of the sample.

Wherein, the sample to be tested is a sample containing the first blood pressure lowering peptide, the second blood pressure lowering peptide, the third blood pressure lowering peptide or the blood pressure lowering protein.

After testing, the IC50 of the sample to be tested is shown in Table 2.

Table 2 IC50 of samples to be tested

blood pressure lowering peptide IC₅₀(μM) First blood pressure lowering peptide (TTW) 0.61 Second blood pressure lowering peptide (VHW) 0.91 Third blood pressure lowering peptide (KAKW) 2.02

The test results show that the three blood pressure-lowering peptides have obvious inhibitory effect on angiotensin converting enzyme in vitro, and its inhibitory effect is close to the in vitro inhibitory effect of the classic drug lisinopril for the treatment of hypertension (IC50 = 0.0214 μM).

<Experiment 2: In vivo inhibition experiment of various blood pressure-lowering peptides on angiotensin-converting enzyme>

This experiment mainly verifies the effect of various blood pressure-lowering peptides on the blood pressure of hypertensive rats, which includes the following steps:

(1) Obtain spontaneously hypertensive rats (SHRs, 10 weeks old, male, body weight 250-320g) with blood pressure over 180mmHg, purchased from Shanghai Slack Animal Experiment Co.;

(2) The hypertensive rats were raised in groups of 6, under the temperature of 22 ± 2 °C for 12 hours, with 12 hours of circular light, and supplied with food and drinking water;

(3) Dissolve different blood pressure-lowering peptides in physiological saline, the experimental group is calculated according to the amount of 3 μmol/Kg of each hypertensive rat, the physiological saline is administered by gavage according to the amount of 1ml/100g rat body weight, and the control group is The same dose of normal saline was administered by gavage while feeding the hypotensive drug lisinopril;

(4) After gavage, the blood pressure of these hypertensive rats was measured at 0h, 1h, 2h, 4h, 6h and 8h respectively. All results were measured in triplicate.

The results are shown in Figure 4. As can be seen from Figure 4, compared with the blank experimental group (control), the blood pressure of the three blood pressure-lowering peptides decreased significantly, indicating that they have a blood pressure lowering effect in the body. At the same time, compared with the lisinopril group, the three blood pressure lowering The blood pressure lowering effect of the peptide group can be considered to be better than that of lisinopril in the early stage.

In conclusion, the blood pressure-lowering peptide not only has good blood pressure-lowering activity in vitro, but also has good in vivo activity after being digested and absorbed by the gastrointestinal tract, and its blood pressure-lowering function is not lost.

The present invention will be further described below with reference to each embodiment.

Example 1: Antihypertensive drug tablet

The hypotensive drug tablet of this embodiment contains 5 wt % of hypotensive peptide and 95 wt % of excipients. The blood pressure lowering peptide is the first blood pressure lowering peptide, and the excipients are starch and magnesium stearate.

In fact, the blood pressure lowering peptide can also be any one or more of the second blood pressure lowering peptide, the third blood pressure lowering peptide and the blood pressure lowering protein. The auxiliary material can be any one or more of starch, sucrose and maltodextrin. The content of the blood pressure lowering peptide may be 1-20 wt % and the content of the adjuvant may be 80 wt-99 wt %.

Example 2: Hypotensive health care products

The hypotensive drug tablet of the present embodiment contains 15 wt % of hypotensive peptide and 85 wt % of excipients. The blood pressure lowering peptide is the third blood pressure lowering peptide, and the excipients are starch, honey, dietary fiber, arginine and glutathione.

Wherein, the blood pressure lowering peptide may also be any one or more of the first blood pressure lowering peptide, the second blood pressure lowering peptide and the blood pressure lowering protein. The content of the blood pressure lowering peptide may be 1-20wt% and the content of the auxiliary material may be 80wt-99wt%.

The foregoing description of the embodiments is provided to facilitate understanding and use of the present invention by those of ordinary skill in the art. It will be apparent to those skilled in the art that various modifications to these embodiments can be readily made, and the generic principles described herein can be applied to other embodiments without inventive step. Therefore, the present invention is not limited to the above-mentioned embodiments, and improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should all fall within the protection scope of the present invention.

sequence listing

<110> East China University of Science and Technology

<120> Blood pressure lowering peptides and blood pressure lowering proteins and their applications

<130> 191034

<141> 2019-04-25

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 4

<212> PRT

<213> Natural Foods (2 Ambystoma laterale x Ambystoma jeffersonianum)

<400> 1

Lys Ala Lys Trp

1

Claims (5)

1. A hypotensive peptide, characterized by: the amino acid sequence is shown as SEQ ID NO: 1 is shown.

2. Use of the antihypertensive peptide according to claim 1 for producing an angiotensin converting enzyme inhibitor.

3. Use of the antihypertensive peptide according to claim 1 for the preparation of a hypotensive drug.

4. The use of the antihypertensive peptide according to claim 1 for the production of a health product for lowering blood pressure.

5. Use of the antihypertensive peptide according to claim 1 for producing foods for lowering blood pressure.

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