CN110540577A - A duck-derived polypeptide with hypotensive effect and application thereof - Google Patents

Abstract

The invention provides a duck-derived polypeptide with hypotensive effect and application thereof, belonging to the field of biotechnology. The amino acid sequence of the duck-derived polypeptide with hypotensive effect is shown in SEQ ID No.1 in the sequence table. The polypeptide has strong ACE enzyme inhibitory activity, therefore, it can be used as an ACE inhibitor to inhibit the activity of angiotensin converting enzyme, and at the same time, ACE enzyme is involved in the occurrence and development of various diseases. Therefore, the duck-derived Application of the polypeptide in the preparation of medicines for treating hypertension, cardiovascular disease, heart failure or renal failure.

Description

A duck-derived polypeptide with hypotensive effect and application thereof

technical field

The invention belongs to the field of biotechnology, and in particular relates to a duck-derived polypeptide with hypotensive effect and application thereof.

Background technique

Angiotensin converting enzyme (Angiotensin converting enzyme, ACE, EC3.4.15.1, once used in the literature as kininaseIl, dipeptidyl carboxypeptidase I, etc.) is a dicarboxypeptidase, which is a key enzyme leading to high blood pressure. It passes Hydrolysis converts angiotensin I to angiotensin II. At the same time, ACE can also inactivate bradykinin, both of which can lead to vasoconstriction, which can cause hypertension. Therefore, ACE is considered to be an important factor causing hypertension. Studies have found that angiotensin-converting enzyme inhibitors (ACEI) can lower blood pressure by inhibiting the activity of ACE. ACE inhibitors are widely used in the treatment of cardiovascular, hypertension, heart failure, renal failure and other diseases. Common antihypertensive drugs include diuretics, tablet receptor blockers, calcium channel blockers, angiotensin-converting enzyme inhibitors, a-receptor blockers, angiotensin II receptor antagonists, etc. These drugs The effect on lowering blood pressure is mixed.

ACE inhibitors were originally discovered from snake venom, and then people found that ACE inhibitory peptides extracted from food materials, such as gelatin, casein, fish, fig gum, α-zein, etc., can be used as raw materials for the preparation of ACE inhibitory peptides. The potential specific target active peptides contained in different peptide sequences are different, and the peptide sequences obtained under the action of the same enzyme or different enzymes are different.

Contents of the invention

In view of this, the purpose of the present invention is to provide a duck-derived polypeptide with ACE inhibitory activity and its application.

The invention provides a duck-derived polypeptide with hypotensive effect, the amino acid sequence of the duck-derived polypeptide is shown in SEQ ID No.1 in the sequence table.

The invention provides an angiotensin-converting enzyme inhibitor, including the duck-derived polypeptide.

The present invention provides the application of the duck-derived polypeptide or the angiotensin-converting enzyme inhibitor in inhibiting the activity of angiotensin-converting enzyme.

Preferably, the concentration of the duck-derived polypeptide is ≥1.0 mg/mL.

Preferably, the concentration of the duck-derived polypeptide is 2.0-3.0 mg/mL.

The invention provides the application of the duck-derived polypeptide or the angiotensin-converting enzyme inhibitor in the preparation of a drug for treating hypertension.

The invention provides the application of the duck-derived polypeptide or the angiotensin-converting enzyme inhibitor in health products for lowering blood pressure.

The invention provides the application of the duck-derived polypeptide or the angiotensin-converting enzyme inhibitor in the preparation of medicines for treating cardiovascular disease, heart failure disease or renal failure disease.

The invention provides a duck-derived polypeptide with hypotensive effect, the amino acid sequence of the duck-derived polypeptide is shown in SEQ ID No.1 in the sequence table. The polypeptide is a 12-peptide sequence and has inhibitory activity against ACE targets. From the results of the ACE inhibitory activity experiment, it can be seen that the ACE inhibitory activity at 1.0 mg/mL is 78.16%, and the ACE inhibitory activity at 2.0 mg/mL is 86.13%, indicating that the polypeptide provided by the present invention has strong ACE enzyme inhibitory activity , the peptide is reported for the first time, and it belongs to a new functional peptide with ACE inhibitory activity, which provides a new treatment method for subsequent research on diseases involving ACE enzymes.

Detailed ways

The present invention provides a duck-derived polypeptide with hypotensive effect, the amino acid sequence of the duck-derived polypeptide is shown in the sequence table SEQ ID No.1 (Gln-Arg-Thr-Tyr-Ala-Ser-Thr-Lys- Glu-Ala-His-Pro). The polypeptide is derived from Chinese duck (NCBI Reference Sequence: NP_001297320.1, Molecular cloning of the perilipin gene and its association with carcass and fattraits in Chineseducks. Genet. Mol. Res. 2013, 12 (2), 1582-1592) protein, expressed The gene of the protein is correlated with the quality of duck meat. Through the computer-aided virtual enzymatic hydrolysis technology, the obtained ACE inhibitory peptide was obtained under the action condition of pepsin (pH≦1.3). Experiments have proved that the duck-derived polypeptide has strong angiotensin-converting enzyme inhibitory activity. Angiotensin-converting enzyme is a key enzyme that causes hypertension. It converts angiotensin I into angiotensin II through hydrolysis. At the same time, ACE can also inactivate bradykinin, both of which can lead to vasoconstriction, thereby causing hypertension, and the duck-derived polypeptide has the effect of lowering blood pressure by inhibiting the activity of ACE. The present invention has no special limitation on the source of the duck-derived polypeptide, and a common polypeptide synthesis method in the art can be used. In the embodiment of the present invention, the duck-derived polypeptide was entrusted to Jill Biochemical (Shanghai) Co., Ltd. to synthesize.

The invention provides an angiotensin-converting enzyme inhibitor, including the duck-derived polypeptide. The mass percent content of the duck-derived polypeptide is preferably 80-90%. The angiotensin-converting enzyme inhibitor preferably also includes adjuvants acceptable in the field of inhibitors. The present invention has no special limitation on the preparation method of the angiotensin-converting enzyme inhibitor, and the preparation method of the enzyme inhibitor known in the art can be adopted.

Based on the strong ACE inhibitory activity of the duck-derived polypeptide, the present invention provides the use of the duck-derived polypeptide or the angiotensin-converting enzyme inhibitor in inhibiting the activity of angiotensin-converting enzyme. In the present invention, the concentration of the duck-derived polypeptide is preferably ≥1.0 mg/mL, more preferably 2.0-3.0 mg/mL. The solvent for dissolving the duck-derived polypeptide is preferably a 0.1 moL/L boric acid buffer containing 0.3 moL/L NaCl and a pH value of 8.3.

Because ACE is a key enzyme leading to high blood pressure, the action of ACE can lead to vasoconstriction, thereby causing high blood pressure. At the same time, based on the strong ACE enzyme inhibitory activity of the duck-derived polypeptide, the present invention provides the duck-derived polypeptide or Application of the angiotensin-converting enzyme inhibitor in the preparation of medicines for treating hypertension. The invention provides the application of the duck-derived polypeptide or the angiotensin-converting enzyme inhibitor in health products for lowering blood pressure.

Based on the fact that ACE inhibitors are also widely used in the treatment of diseases such as cardiovascular disease, heart failure, and renal failure, the present invention provides that the duck-derived polypeptide or the angiotensin-converting enzyme inhibitor can be used in the treatment of cardiovascular diseases, heart failure diseases, etc. or the application of drugs in renal failure diseases.

In the present invention, the dosage form of the medicine prepared by the duck-derived polypeptide is preferably an oral dosage form. In the medicament, the concentration of the duck-derived polypeptide is 1.0-2.0 mg/mL, more preferably 2.0 mg/mL. The method of taking the medicine is preferably 2 g each time, 2 to 3 times a day.

A duck-derived polypeptide with hypotensive effect and its application provided by the present invention will be described in detail below in conjunction with the examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

ACE inhibitory activity detection method

ACE catalyzes the decomposition of angiotensin I mimic Hippuryl-L-Histidyl-L-Leucine (HHL) at 37°C and a pH value of 8.3 to produce hippuric acid (HA), which has a characteristic absorption peak at 225nm in ultraviolet light When adding ACE inhibitors, the catalytic decomposition of ACE to HHL is inhibited, and the generation of hippuric acid is reduced. By HPLC method, the change of the amount of hippuric acid generated before and after adding the inhibitor can be calculated to calculate the size of the inhibitory activity.

The reaction system is as follows: add 20 μL 0.1U/mL ACE and 50 μL 1.0 mg/mL ACE inhibitory peptide solution in sequence, incubate at 37°C for 5 minutes, then add 10 μL 5mM HHL substrate to start the catalytic reaction of ACE, shake the water bath at 37°C for 30 minutes The reaction was terminated by adding 250 μL of 1.0 mol/L HCl. After the system solution was passed through a 0.45 μm filter membrane, the content of hippuric acid (HA) was detected and analyzed by RP-HPLC. Under the same conditions as above, 50 μL of 0.1 mol/L boric acid buffer solution (containing 0.3 mol/L NaCl, pH=8.3) was used instead of ACE inhibitor as a blank reaction system. The above-mentioned ACE and HHL substrates all use 0.1moL/L borate buffer solution (containing 0.3moL/L NaCl, pH=8.3) as a solvent. ACE inhibitory peptide (KDTISTP) was dissolved in 0.1moL/L borate buffer (containing 0.3moL/L NaCl, pH=8.3) to obtain ACE inhibitory peptide solution.

HPLC chromatographic conditions: Solvent I is 0.05% trifluoroacetic acid (TFA) and 0.05% triethylamine (TTA) dissolved in deionized water (that is, every liter of solvent I contains 0.5mL of trifluoroacetic acid and 0.5mL of triethylamine Amine), solvent II is 100% chromatographically pure acetonitrile. The ratio of solvent Ⅰ to solvent Ⅱ is 70%: 30% (volume ratio), the ultimate3000 Diane liquid chromatograph, the chromatographic column is waters Symmetry C185μm4.6×250mm, the flow rate is 0.5mL/min, the injection volume is 10μL, and the detection The wavelength is 225nm, and the detection column temperature is 30°C.

RP-HPLC detection: solvent I is 0.05% (V/V) trifluoroacetic acid (TFA) and 0.05% (v/v) triethylamine (TTA) dissolved in deionized water, solvent II is 100% chromatographically pure B eye. The ratio of solvent I to solvent II is 70%:30% (volume ratio), the flow rate is 0.5mL/min, the detection wavelength is 225nm, and the detection column temperature is 30°C.

ACE inhibitory activity was calculated according to the following formula:

I%=(A-B)/A×100%

A: The peak area of hippuric acid without adding short peptide inhibitors;

B: The peak area of hippuric acid when adding short peptide inhibitors;

ACE: 1 U unit is defined as the amount of ACE consumed to produce 1 μM hippuric acid by catalyzing the substrate (Hippuryl-L-Histidyl-L-Leucine, HHL) within 1 minute at 37°C under standard detection conditions. That is, the active unit of ACE.

Results: The ACE inhibitory activity of peptide KDTISTP at 1.0 mg/mL was 78.16%.

Example 2

ACE inhibitory activity detection method

ACE catalyzes the decomposition of angiotensin I mimic Hippuryl-L-Histidyl-L-Leucine (HHL) at 37°C and a pH value of 8.3 to produce hippuric acid (HA), which has a characteristic absorption peak at 225nm in ultraviolet light When adding ACE inhibitors, the catalytic decomposition of ACE to HHL is inhibited, and the generation of hippuric acid is reduced. By HPLC method, the change of the amount of hippuric acid generated before and after adding the inhibitor can be calculated to calculate the size of the inhibitory activity.

The reaction system is as follows: add 20 μL 0.1U/mL ACE and 50 μL 2.0 mg/mL ACE inhibitory peptide solution in sequence, incubate at 37°C for 5 minutes, then add 10 μL 5mM HHL substrate to start the catalytic reaction of ACE, shake the water bath at 37°C for 30 minutes The reaction was terminated by adding 250 μL of 1.0 mol/L HCl. After the system solution was passed through a 0.45 μm filter membrane, the content of hippuric acid (HA) was detected and analyzed by RP-HPLC. Under the same conditions as above, 50 μL of 0.1 mol/L boric acid buffer solution (containing 0.3 mol/L NaCl, pH=8.3) was used instead of ACE inhibitor as a blank reaction system. The above-mentioned ACE and HHL substrates all use 0.1moL/L borate buffer solution (containing 0.3moL/L NaCl, pH=8.3) as a solvent. ACE inhibitory peptide (KDTISTP) was dissolved in 0.1moL/L borate buffer (containing 0.3moL/L NaCl, pH=8.3) to obtain ACE inhibitory peptide solution.

HPLC chromatographic conditions: Solvent I is 0.05% trifluoroacetic acid (TFA) and 0.05% triethylamine (TTA) dissolved in deionized water (that is, every liter of solvent I contains 0.5mL of trifluoroacetic acid and 0.5mL of triethylamine Amine), solvent II is 100% chromatographically pure acetonitrile. The ratio of solvent Ⅰ to solvent Ⅱ is 70%: 30% (volume ratio), the ultimate3000 Diane liquid chromatograph, the chromatographic column is waters Symmetry C185μm4.6×250mm, the flow rate is 0.5mL/min, the injection volume is 10μL, and the detection The wavelength is 225nm, and the detection column temperature is 30°C.

RP-HPLC detection: solvent I is 0.05% (V/V) trifluoroacetic acid (TFA) and 0.05% (v/v) triethylamine (TTA) dissolved in deionized water, solvent II is 100% chromatographically pure B eye. The ratio of solvent I to solvent II is 70%:30% (volume ratio), the flow rate is 0.5mL/min, the detection wavelength is 225nm, and the detection column temperature is 30°C.

ACE inhibitory activity was calculated according to the following formula:

I%=(A-B)/A×100%

A: The peak area of hippuric acid without adding short peptide inhibitors;

B: The peak area of hippuric acid when adding short peptide inhibitors;

ACE: 1 U unit is defined as the amount of ACE consumed to produce 1 μM hippuric acid by catalyzing the substrate (Hippuryl-L-Histidyl-L-Leucine, HHL) within 1 minute at 37°C under standard detection conditions. That is, the active unit of ACE.

Results: The ACE inhibitory activity of peptide KDTISTP at 1.0 mg/mL was 86.13%.

It can be seen from the above examples that there is a dose-effect relationship between the activity and concentration of the duck-derived polypeptide sequence provided by the present invention. The polypeptide has ACE inhibitory activity which has not been reported, and belongs to a new functional peptide with ACE inhibitory activity.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Zhejiang Academy of Agricultural Sciences

<120> A duck-derived polypeptide with hypotensive effect and its application

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 12

<212> PRT

<213> Artificial Sequence

<400> 1

Gln Arg Thr Tyr Ala Ser Thr Lys Glu Ala His Pro

1 5 10

Claims (8)

1. The duck-origin polypeptide with the blood pressure lowering effect is characterized in that the amino acid sequence of the duck-origin polypeptide is shown in a sequence table SEQ ID No. 1.

2. an angiotensin converting enzyme inhibitor comprising the duck-origin polypeptide of claim 1.

3. use of the duck-derived polypeptide of claim 1 or the angiotensin-converting enzyme inhibitor of claim 2 for inhibiting angiotensin-converting enzyme activity.

4. The use of claim 3, wherein the concentration of said duck-derived polypeptide is greater than or equal to 1.0 mg/mL.

5. The use of claim 3, wherein the concentration of the duck-origin polypeptide is 2.0-3.0 mg/mL.

6. Use of the duck-origin polypeptide of claim 1 or the angiotensin-converting enzyme inhibitor of claim 2 for the preparation of a medicament for the treatment of hypertension.

7. Use of the duck-origin polypeptide of claim 1 or the angiotensin-converting enzyme inhibitor of claim 2 in a health product for lowering blood pressure.

8. Use of the duck-origin polypeptide of claim 1 or the angiotensin-converting enzyme inhibitor of claim 2 for the manufacture of a medicament for the treatment of cardiovascular disease, heart failure disease or renal failure disease.