CN112661811B

CN112661811B - Antihypertensive peptide, long-acting antihypertensive peptide and preparation method thereof

Info

Publication number: CN112661811B
Application number: CN202110079190.0A
Authority: CN
Inventors: 吕应年; 戚怡; 罗辉; 唐晓宁; 林芊杏; 刘欢; 黄细香; 蔡晓璇
Original assignee: Guangdong Medical University; Southern Marine Science and Engineering Guangdong Laboratory Zhanjiang
Current assignee: Guangdong Medical University; Southern Marine Science and Engineering Guangdong Laboratory Zhanjiang
Priority date: 2021-01-21
Filing date: 2021-01-21
Publication date: 2022-10-21
Anticipated expiration: 2041-01-21
Also published as: CN112661811A; WO2022156190A1

Abstract

The invention provides antihypertensive peptide, long-acting antihypertensive peptide and a preparation method thereof, and relates to the technical field of antihypertensive drugs. The antihypertensive peptide of the invention has at least one polypeptide sequence shown in SEQ ID NO.1-SEQ ID NO. 6. The polypeptide is obtained by separating and purifying sandworms for the first time, and experimental results show that the polypeptide has a good blood pressure reducing effect and can be used for preparing blood pressure reducing medicines. The long-acting antihypertensive peptide is polypeptide modified by polyethylene glycol, and can prolong the drug effect time of the polypeptide.

Description

Antihypertensive peptide, long-acting antihypertensive peptide and preparation method thereof

Technical Field

The invention relates to the technical field of antihypertensive drugs, and in particular relates to antihypertensive peptide, long-acting antihypertensive peptide and a preparation method thereof.

Background

Angiotensin converting enzyme inhibitory peptides (angiotensins converting enzyme inhibitory peptides) are polypeptide substances having an inhibitory effect on the activity of Angiotensin Converting Enzyme (ACE). Angiotensin converting enzyme inhibitory peptides generally consist of 2 to 20 amino acid residues, are usually obtained by proteolysis, and achieve the effect of lowering blood pressure by inhibiting the activity of angiotensin converting enzyme, inhibiting the production of angiotensin ii and inhibiting the decomposition of bradykinin. Because the enzyme cutting site of the protease is not fixed, the polypeptide obtained after enzyme cutting is also diversified, and the later screening, separation and purification become the technical difficulties.

Disclosure of Invention

In view of the above, there is a need to provide a antihypertensive peptide, which is a small molecular biological peptide having an antihypertensive effect, can be used for preventing and treating hypertension, and has low side effects and a good antihypertensive effect.

An antihypertensive peptide comprising at least one of the following polypeptide sequences:

Gly-Phe-Ala-Gly-Asp-Asp-Ala-Pro-Arg(SEQ ID NO.1)，

Gly-Lue-Gly-Gly-Lue-Ser-Pro-Glu-Lys(SEQ ID NO.2)，

Lue-Pro-Lys(SEQ ID NO.3)，

Pro-Arg-Pro(SEQ ID NO.4)，

Ser-Arg-Pro(SEQ ID NO.5)，

Arg-Pro-Ala(SEQ ID NO.6)。

the antihypertensive peptide is obtained by separating and purifying marine organism sandworms for the first time, and experiments prove that the antihypertensive peptide has a better antihypertensive effect and can be used for preventing and treating hypertension.

In one embodiment, the antihypertensive peptide has the polypeptide sequence shown in SEQ ID NO. 5. The polypeptide of the sequence has the best antihypertensive effect among 6 polypeptides.

The invention also provides a preparation method of the antihypertensive peptide, which comprises the following steps:

s1, selecting sandworms, and adding alkali liquor to adjust the pH value to 7.5-8.5;

s2, adding enzyme for enzymolysis, heating to inactivate the enzyme after the enzymolysis is finished, and obtaining an enzymolysis mixture;

s3, carrying out solid-liquid separation on the enzymolysis mixture, and reserving liquid to obtain enzymolysis liquid;

s4, performing ultrafiltration dialysis on the enzymatic hydrolysate, and reserving ultrafiltrate with the molecular weight less than or equal to 100 KDa;

s5, separating and purifying the ultrafiltrate to obtain the polypeptide shown as SEQ ID NO.1-SEQ ID NO. 6.

The preparation method can stably and accurately cut the enzyme to obtain the antihypertensive peptide, the antihypertensive peptide is obtained by separating and purifying the antihypertensive peptide from sandworms for the first time, and experiments prove that the polypeptide has the antihypertensive effect.

The activity of angiotensin converting enzyme inhibitory peptides depends on the amino acid type, sequence and spatial structure of the polypeptide. In the enzymolysis process, protein macromolecules are firstly cut into polypeptide fragments by protease, and the polypeptides are further subjected to enzymolysis and cutting to form free amino acids along with the deepening of the enzymolysis degree. Therefore, the enzymolysis process parameters (including the type, the dosage, the pH value, the temperature and the enzymolysis time of the enzyme) are well controlled, the cutting site and the enzymolysis degree can be controlled, and the size and the structure of the polypeptide fragment are determined.

In one embodiment, the step S1 specifically includes: cleaning sandworm, mincing, homogenizing, adding 10-15 times of water, mixing, adding alkali solution to adjust pH to 7.5-8.5.

In one embodiment, the step S2 specifically includes: adding enzyme powder slurry according to 0.5-1% of the sandworm weight, wherein the enzymolysis temperature is 45 +/-5 ℃, the enzymolysis time is 4-6h, after the enzymolysis is finished, heating to 95 +/-5 ℃, and preserving the heat for 3-8 min to inactivate the enzyme, thereby obtaining an enzymolysis mixture.

In one embodiment, the enzyme powder slurry contains trypsin and neutral protease, and the weight ratio of the trypsin to the neutral protease is (2-4): 1.

In one embodiment, the dosage ratio of the sandworms to the enzymes is 100g:0.5-1g. In the enzymolysis process of sandworm (sipunculus nudus) homogenate, if the enzymolysis is insufficient, sipunculus nudus protein exists in a macromolecular state and cannot be cut to obtain a peptide segment with ACE inhibitory activity; if the enzymolysis is excessive, the peptide segment is further cut to form free amino acid without an active structure, so that the ACE inhibitory activity is lost.

In one embodiment, the step S3 specifically includes: and centrifuging the enzymolysis mixture, discarding the precipitate, and retaining the supernatant to obtain an enzymolysis liquid.

In one embodiment, the step S5 specifically includes: loading polyethylene glycol modified copper ion chelating agarose into a column, washing with water, eluting with a phosphate buffer solution until the copper ion chelating agarose is balanced, and performing column chromatography on the ultrafiltrate to obtain the antihypertensive peptide.

The invention also provides a preparation method of the polyethylene glycol modified antihypertensive peptide, which comprises the following steps:

1) Dissolving the antihypertensive peptide according to any one of claims 1 to 2 in a buffer to obtain a polypeptide solution;

2) Mixing polyethylene glycol and glycine, and reacting to obtain activated polyethylene glycol;

3) Mixing the polypeptide solution with activated polyethylene glycol, adding acid to adjust the pH value to 2-4 after the reaction is finished, and stopping the reaction;

4) Dialyzing the product obtained in the step 3), and separating and purifying to obtain the polyethylene glycol modified antihypertensive peptide.

In one embodiment, the step 1) specifically includes: dissolving the antihypertensive peptide in Tris-HCl buffer solution to prepare peptide solution with the concentration of 5-50mg/ml, and cooling to 2-8 ℃ for later use.

In one embodiment, the step 2) is specifically: mixing polyethylene glycol and amino acid, reacting and dehydrating under acidic condition to obtain activated polyethylene glycol, dissolving in acetonitrile-water solution with activated polyethylene glycol concentration of 50-150mg/ml, and cooling to 2-8 deg.C for use.

Preferably, the polyethylene glycol has a molecular weight of 3000-30000. Including but not limited to branched or straight chain polyethylene glycols.

Preferably, the amino acid is glycine. The glycine has small molecular weight and good stability, the activation yield is up to 70 percent, and the activation yield of other amino acids is about 30 to 50 percent. The glycine is adopted for activation, so that the yield is higher, and the byproducts are less.

In one embodiment, the step 3) is specifically: mixing the peptide solution and the activated polyethylene glycol solution according to the proportion of 1 (10-50), and stirring for 36-72h at the temperature of 2-8 ℃; after the reaction is finished, adding acid to adjust the pH value to 2-4, and stopping the reaction.

The hydroxyl group of polyethylene glycol is bonded with the carboxyl group of a single amino acid such as glycine to form an ester bond under acidic conditions to give NH having an amino group ₂ Activated polyethylene glycol at the end. The activated polyethylene glycol and the carboxyl of the small molecular peptide are subjected to dehydration condensation reaction to generate amido bond, namely, the polyethylene glycol is lengthened on the peptide molecule to obtain the antihypertensive peptide modified by the polyethylene glycol.

In one embodiment, the step 4) is specifically: dialyzing the product obtained in the step 3) by using a dialysis membrane with the molecular weight cutoff of 3000Da, adding solid PEG6000 into the dialysis membrane to cover and dehydrate, concentrating, performing gel column chromatography, and eluting by using acetate buffer solution to obtain the polyethylene glycol modified antihypertensive peptide.

The invention also provides the polyethylene glycol modified antihypertensive peptide prepared by the preparation method.

The invention also provides application of the antihypertensive peptide in preparing antihypertensive drugs.

Because the biological products of the polypeptides are easy to be damaged in the digestive tract by oral administration and the degradation activity of the antihypertensive peptides disappears, the biological products can be prepared into injections for injection administration. The administration frequency can be prolonged to once a week or once a month, so that the drug effect and the treatment compliance of the drug are improved, and the pain of patients is relieved.

The invention also provides application of the antihypertensive peptide modified by polyethylene glycol in preparing antihypertensive drugs.

The polyethylene glycol is adopted to modify the small molecular peptide, so that the duration of the drug effect of the antihypertensive peptide can be prolonged.

Compared with the prior art, the invention has the following beneficial effects:

the antihypertensive peptide is obtained by separating and purifying marine organism sandworms for the first time, and experiments prove that the antihypertensive peptide has a good antihypertensive effect and can be used for preventing and treating hypertension. By adopting the preparation method, the antihypertensive peptide can be obtained by separation, the preparation result is stable and reliable, and the separation and purification efficiency is high. The antihypertensive peptide modified by polyethylene glycol can prolong the drug effect time of the polypeptide.

Drawings

FIG. 1 is an MS map of a polypeptide represented by SEQ ID NO. 1;

FIG. 2 is an HPLC chromatogram of the polypeptide shown in SEQ ID NO. 1;

FIG. 3 is an MS map of the polypeptide shown in SEQ ID NO. 2;

FIG. 4 is an HPLC chromatogram of the polypeptide shown in SEQ ID NO. 2;

FIG. 5 is an MS map of the polypeptide represented by SEQ ID NO. 3;

FIG. 6 is an HPLC chromatogram of the polypeptide represented by SEQ ID NO. 3;

FIG. 7 is an MS map of the polypeptide represented by SEQ ID NO. 4;

FIG. 8 is an HPLC chromatogram of the polypeptide represented by SEQ ID NO. 4;

FIG. 9 is an MS map of the polypeptide represented by SEQ ID NO. 5;

FIG. 10 is an HPLC chromatogram of the polypeptide shown in SEQ ID NO. 5;

FIG. 11 is an MS map of the polypeptide represented by SEQ ID NO. 6;

FIG. 12 is an HPLC chromatogram of the polypeptide represented by SEQ ID NO. 6;

FIG. 13 shows ACE inhibition of 6 polypeptides (10 mmol/L);

FIG. 14 is the ACE inhibitory activity of 6 polypeptidesIC ₅₀ And (5) value test results.

Detailed Description

In order that the invention may be more fully understood, preferred embodiments will now be given. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Example 1

The enzymolysis extraction and purification of the antihypertensive peptide comprises the following steps:

(1) Cleaning sandworm until no sand exists, and mincing and homogenizing;

(2) Adding pure water with the weight being 12 times that of the sandworms, uniformly mixing, and adding 1% of sodium hydroxide solution to adjust the pH value to 7.5;

(3) Mixing trypsin and neutral protease in a weight ratio of 3:1, adding water to dissolve the mixture into slurry, adding enzyme powder slurry according to 0.8 percent of the weight of the sandworm, wherein the dosage ratio of the sandworm to the enzyme is 100g:0.7g;

(4) Slowly adding the enzyme powder slurry into the sandworm liquid while stirring, uniformly mixing, setting the temperature at 45 ℃, and performing enzymolysis for 5 hours;

(5) After enzymolysis, heating to 95 ℃, and preserving heat for 5min to kill enzyme;

(6) Centrifuging with a low-temperature high-speed centrifuge, separating solid and liquid, removing precipitate, and collecting supernatant;

(7) Carrying out ultrafiltration dialysis on the supernatant, and reserving ultrafiltrate with the molecular weight less than or equal to 100 KDa;

(8) Loading polyethylene glycol modified metal chelate affinity chromatography medium (polyethylene glycol modified copper ion Chelating Sepharose Fast Flow, mPEG-CSFF-Cu) into column, loading column by natural sedimentation method with column volume of about 20ml, eluting with distilled water to remove unbound metal ions, and eluting with pH6.8 sodium phosphate buffer solution (containing 0.5mol/L sodium chloride) to balance; loading, wherein the loading buffer solution is PH6.8 sodium phosphate buffer solution (containing 0.5mol/L sodium chloride), the elution buffer solution is citric acid buffer solution with pH5.0, and performing metal chelating chromatographic column chromatography to obtain the following polypeptides:

TABLE 1 polypeptide sequences

Serial number	Sequence of	Sequence shorthand	Name(s)
				SEQ ID NO.1	Gly-Phe-Ala-Gly-Asp-Asp-Ala-Pro-Arg	GFAGDDAPR	S-4521
SEQ ID NO.2	Gly-Lue-Gly-Gly-Lue-Ser-Pro-Glu-Lys	GLGGLSPEK	S-4522
				SEQ ID NO.3	Lue-Pro-Lys	LPK	S-4523
SEQ ID NO.4	Pro-Arg-Pro	PRP	S-4524
				SEQ ID NO.5	Ser-Arg-Pro	SRP	S-4525
SEQ ID NO.6	Arg-Pro-Ala	RPA	S-4526

(9) And (3) dialyzing, desalting, concentrating, vacuum freeze-drying and storing the small-molecular peptide liquid.

The mass spectrum and HPLC results of the polypeptides obtained in this example are shown in FIGS. 1 to 12. The results of HPLC testing of the polypeptides S-4521 to S-4526 are shown in tables 2 to 7, respectively.

TABLE 2.S-4521 HPLC assay results

TABLE 3.S-4522 HPLC assay results

TABLE 4.S-4523 HPLC assay results

TABLE 5.S-4524 HPLC assay results

TABLE 6.S-4525 HPLC assay results

TABLE 7.S-4526 HPLC assay results

Example 2

Preparing the polyethylene glycol modified antihypertensive peptide.

(1) Dissolving the small molecular peptide obtained in the example 1 in a Tris-HC buffer solution with the pH value of 9.0 to prepare a peptide solution with the concentration of 20mg/ml, and cooling to 4 ℃;

(2) Mixing polyethylene glycol (molecular weight 4000) and glycine according to a molar ratio of 1:1, reacting under an acidic condition to obtain activated polyethylene glycol, dissolving the activated polyethylene glycol in an acetonitrile-water solution (the volume ratio of acetonitrile to water is 98);

(3) Mixing the peptide solution and the activated polyethylene glycol solution according to a volume ratio of 1;

(4) Bagging and dialyzing the product obtained in the step (3) by using a 3000Da dialysis membrane, removing unreacted small molecular components, putting the dialysis membrane into an aluminum box, storing in a refrigerator, adding a solid PEG6000 to cover and dehydrate, and concentrating to the volume of 1/5;

(5) And (3) loading the concentrated sample to a gel column for chromatography, eluting with acetate buffer solution, collecting elution peaks in a segmented manner according to ultraviolet absorption, and determining the sample with the purity of more than 90% by electrophoretic detection.

Experimental example 1

1. ACE inhibitory activity test.

Six polypeptides separated in example 1 were subjected to ACE inhibitory activity test as follows:

the six polypeptides are respectively prepared into 10mmol/L peptide solution, and the ACE inhibitory activity is determined by adopting a classical hippuric acid content method.

The test results are shown in fig. 13. From the results, it can be seen that the above polypeptides all have ACE inhibitory activity, with S4525 being the most effective.

2. ACE inhibitory Activity IC ₅₀ And (5) testing the value.

The six polypeptides are respectively prepared into different concentrations, and the ACE inhibitory activity IC of each polypeptide is tested by the same method ₅₀ The value is obtained.

The results are shown in FIG. 14 and Table 8. From the results, it can be seen that S4525 corresponds to IC ₅₀ The lowest value (30.5. Mu. Mol/L).

TABLE 8 ACE inhibitory IC of polypeptides ₅₀ Value of

Polypeptides	IC ₅₀ (mmol/L)
		S-4521	0.858
S-4522	0.906
		S-4523	13.34
S-4524	0.465
		S-4525	0.0305
S-4526	0.961

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent should be subject to the appended claims.

Sequence listing

<110> southern China laboratory of Guangdong province in ocean science and engineering (Zhanjiang)

Guangdong Medical University

<120> antihypertensive peptide, long-acting antihypertensive peptide and preparation method thereof

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9

<212> PRT

<213> Sipunculus nudus

<400> 1

Gly Phe Ala Gly Asp Asp Ala Pro Arg

1 5

<210> 2

<211> 9

<212> PRT

<213> Sipunculus nudus

<400> 2

Gly Leu Gly Gly Leu Ser Pro Glu Lys

1 5

<210> 3

<211> 3

<212> PRT

<213> Sipunculus nudus

<400> 3

Leu Pro Lys

1

<210> 4

<211> 3

<212> PRT

<213> Sipunculus nudus

<400> 4

Pro Arg Pro

1

<210> 5

<211> 3

<212> PRT

<213> Sipunculus nudus

<400> 5

Ser Arg Pro

1

<210> 6

<211> 3

<212> PRT

<213> Sipunculus nudus

<400> 6

Arg Pro Ala

1

Claims

1. The application of the antihypertensive peptide in preparing the antihypertensive drug is characterized in that the polypeptide sequence of the antihypertensive peptide is as follows: ser-Arg-Pro.