CN111004308A - Heptapeptide for inhibiting angiotensin converting enzyme and application thereof - Google Patents
Heptapeptide for inhibiting angiotensin converting enzyme and application thereof Download PDFInfo
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- CN111004308A CN111004308A CN201911393864.3A CN201911393864A CN111004308A CN 111004308 A CN111004308 A CN 111004308A CN 201911393864 A CN201911393864 A CN 201911393864A CN 111004308 A CN111004308 A CN 111004308A
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- heptapeptide
- ace
- inhibitory activity
- angiotensin converting
- ace inhibitory
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Images
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Engineering & Computer Science (AREA)
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- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Heart & Thoracic Surgery (AREA)
- Pharmacology & Pharmacy (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Cardiology (AREA)
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- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a heptapeptide for inhibiting angiotensin converting enzyme and application thereof. The polypeptide comprises 7 amino acid residues, has the molecular weight of 818.12, the theoretical isoelectric point of 5.49 and the amino acid sequence of: valine-isoleucine-proline-valine-proline-phenylalanine (Val-Ile-Pro-Val-Pro-Phe). The heptapeptide is derived from an edible yeast enzymolysis product and can be prepared by solid phase synthesis. The polypeptide of the invention has strong Angiotensin Converting Enzyme (ACE) inhibitory activity and half inhibitory concentration IC to ACE50Is 16.43 mu M(ii) a And the compound has small influence on cell proliferation, low cytotoxicity and high safety, can be used for developing and preparing blood pressure lowering functional foods or medicines, and has good application prospect.
Description
Technical Field
The invention belongs to the field of functional foods and biomedicines, and particularly relates to a heptapeptide for inhibiting angiotensin converting enzyme and application thereof.
Background
Hypertension is one of the diseases with the highest mortality in the world, and attracts people's attention. Among the factors regulating human blood pressure, Angiotensin Converting Enzyme (ACE) is the main factor affecting the balance of two systems of pressure raising and lowering, and thus becomes an ideal target for treating diseases such as hypertension and heart failure. Inhibiting ACE activity, and effectively lowering blood pressure.
The artificially synthesized antihypertensive drug has good antihypertensive effect, but the side effect is not negligible. Such as cough, taste dysfunction, allergy and hypotension.
Therefore, research and development of ACE inhibitors with better antihypertensive effect and lower toxic and side effects have urgent needs for prevention and treatment of hypertension diseases.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the heptapeptide with ACE (angiotensin converting enzyme) inhibitory activity, wherein the heptapeptide is derived from a natural product, the invention utilizes edible yeast to hydrolyze by using a bacillus subtilis fermentation enzyme preparation with protease and β -glucanase activity, and yeast enzymolysis products are obtained by LC-MS/MS identification and screening, and researches show that the heptapeptide has strong inhibitory activity on ACE.
It is another object of the present invention to provide the use of said heptapeptide.
Still another object of the present invention is to provide a functional food for lowering blood pressure, an ACE inhibitor or a blood pressure lowering drug.
The purpose of the invention is realized by the following technical scheme:
a heptapeptide with ACE inhibitory activity has an amino acid sequence of valine-isoleucine-proline-valine-proline-phenylalanine (Val-Ile-Pro-Val-Pro-Phe-Phe).
The heptapeptide having ACE inhibitory activity can be prepared by means of a technique conventionally used in the art, for example, by solid phase synthesis.
The application of the heptapeptide with ACE inhibitory activity in preparing an ACE inhibitor.
The heptapeptide with ACE inhibitory activity is applied to a blood pressure lowering functional food.
The heptapeptide with ACE inhibitory activity is applied to the preparation of the antihypertensive drug.
A functional food for lowering blood pressure contains the heptapeptide with ACE inhibitory activity.
An ACE inhibitor contains the heptapeptide with ACE inhibitory activity.
A hypotensive agent contains the heptapeptide having ACE inhibitory activity.
Compared with the prior art, the invention has the following advantages and effects:
1. the heptapeptide has good ACE inhibitory activity, and has half-inhibition rate IC to ACE5016.43. mu.M.
2. The method is a micromolecular polypeptide, the structure is easy to regulate and control, and the micromolecular polypeptide is easy to synthesize and modify so as to obtain better activity and has obvious application potential.
3. The heptapeptide is derived from an enzymolysis product of edible yeast, and the edible yeast is listed as a Generally Recognized As Safe (GRAS) food ingredient released by FDA, so that the safety is high.
Drawings
FIG. 1 is a chromatogram of a solid phase synthesized heptapeptide VIPVPFF.
FIG. 2 is a mass spectrum of a solid phase synthesized heptapeptide VIPVPFF.
Fig. 3 is a graph analyzing the results of inhibition of ACE by different concentrations of vivvpff.
FIG. 4 is a Lineweaver-Burk double reciprocal plot of the polypeptide VIPVPFF against ACE.
FIG. 5 is a graph showing the analysis of the effect of the VIPVPFF polypeptide on the proliferation of human umbilical vein endothelial cells; wherein # and # indicate that the sample group was significantly different from the control group (p <0.01) after 24 hours and 48 hours of incubation, respectively, and # indicates that the sample group was significantly different from the control group (p <0.05) after 24 hours of incubation.
FIG. 6 is a graph of the ACE inhibitory activity of a synthetic polypeptide derived from yeast zymolyte.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
The bacillus subtilis HU528 in this embodiment is stored in the Guangdong province microbial strain storage center of the Guangzhou microbial research institute of No. 59 building and No. 5 building of the Middleya Zhonglu 100, Guangzhou city, with the storage number being GDMCC NO: 60364, which is disclosed in chinese patent application CN 201810668986.8.
1. Preparation of enzyme preparations
The liquid culture medium was prepared as follows: adding peptone 1.5% (w/w, the same below), glucose 0.8%, CaCl in distilled water20.05 percent and 0.15 percent of NaCl, and after being uniformly mixed, the pH value of the liquid culture medium is adjusted to 6.0. Sterilizing at 121 ℃ for 20min, inoculating bacillus subtilis HU528 in logarithmic growth phase into a liquid culture medium according to 1.0% (v/v), fermenting and culturing for 60h under the conditions that the temperature is 25 ℃ and the stirring speed is 180r/min, centrifuging the obtained fermentation liquor at 4 ℃ and 8000r/m for 10min, and removing thalli, wherein the fermentation supernatant is the enzyme preparation.
2. Determination of protease Activity
The protease activity of the obtained enzyme preparation was measured.
The protease activity is determined according to the national standard GB/T23527-2009, tyrosine with different concentrations is used as a standard curve, and the detailed operation is as follows. Diluting the enzyme solution to 1mL by using a phosphate buffer solution with pH 7.5, preheating 2% (w/v) casein (pH 7.5) to 1mL in a water bath at 40 ℃, uniformly mixing, placing in the water bath at 40 ℃ for reaction for 10min, adding 2mL of 0.4mol/L trichloroacetic acid to stop the reaction after the hydrolysis reaction is finished, centrifuging at 10000g for 5min after the water bath at 40 ℃ is 20min, and discarding the precipitate; taking 1mL of supernatant, adding 1mL of forlin phenol reagent and 5mL of 0.4mol/L Na2CO3Mixing, keeping at 40 deg.C for 20min, and measuring light absorption value at 680 nm. In the blank control group, 1mL of enzyme solution was added with 2mL of 0.4mol/L trichloroacetic acid, reacted for 10min, and then 1mL of 2% (w/v) casein was added, followed by the same treatment as in the experimental group. Each measurement was repeated three times and averaged. The measured OD was passed through a standard curve to obtain the corresponding tyrosine concentration, and the enzyme activity was calculated.Protease activity unit definition: in a certain temperature and reaction system, 1mL of sample diluted by a certain multiple hydrolyzes casein per minute to generate 1 mu g of tyrosine, which is 1 protease activity unit, and the enzyme activity per milliliter of enzyme solution is expressed by U/mL. And measuring the activity of the protease in the enzyme preparation to be 1600U/mL.
3, β -measurement of dextranase Activity
The β -glucanase activity of the resulting enzyme preparation was determined.
β -glucanase activity is measured by DNS colorimetric method, 0.1mL, 0.2mL, 0.3mL, 0.4mL and 0.5mL of glucose standard solution (1mg/mL) are respectively absorbed, distilled water is supplemented to 1mL, 1mL of distilled water is used as blank control, 3mL of DNS reagent is added into each test tube, after shaking up, boiling water bath is carried out for 5min, the absorbance value at 540nm is measured after cooling down at normal temperature, a standard curve is drawn, 0.5mL of the enzyme preparation diluted by a certain multiple is taken, 0.5mL of soluble yeast β -glucan solution (1%, w/v) preheated for 3min at 55 ℃ is added, after shaking up, the reaction is carried out for 10min at 55 ℃, 3mL of DNS solution is immediately added after the reaction is completed, after fully mixing, boiling water bath is carried out for 5min, the absorbance value at 540nm is measured after cooling down at normal temperature, meanwhile, 0.5mL of soluble yeast β -glucan solution which is firstly boiled and inactivated is taken as blank yeast solution, the calculation of the enzyme activity is carried out according to the formula (1).
β glucanase activity unit (U/mL) ═ CxK)/(180 xT) (equation 1)
In the formula, C: standard glucose concentration (μ g/mL) corresponding to the absorbance value measured on the standard curve; k: dilution times; 180: the molecular weight of glucose; t: reaction time, 10 min.
β -glucanase activity is defined as that under proper reaction conditions, the amount of enzyme protein needed for decomposing β -glucan to produce 1 mu mol of glucose per minute is defined as one enzyme activity unit, and the enzyme activity per mL of enzyme solution is expressed as U/mL, and the β -glucanase activity in the enzyme preparation is measured to be 3.0U/mL.
4. Preparation and research of yeast enzymolysis product
Adding 9600U of the enzyme preparation (6mL of the enzyme preparation/g of the dry yeast) containing protease activity into edible yeast powder (from Wuzhou pharmaceutical Co., Ltd., Guangdong, dried yeast, 6 months 2017) per gram of the yeast powder, and supplementing sterile water until the volume ratio (material-liquid ratio, w/v) of the edible yeast powder to the mixed solution is 1: 25. The pH of the mixture was adjusted to 9.0. Performing enzymolysis reaction at 70 deg.C for 2 hr, inactivating enzyme at 100 deg.C for 10min, centrifuging to obtain supernatant, and spray drying to obtain yeast enzymolysis product. The conditions of spray drying are that the air inlet temperature is 150 ℃, the air outlet temperature is 80 ℃, and the feeding flow is 25 mL/min.
The above yeast enzymatic hydrolysate was identified by LC-MS/MS, and it was found that a heptapeptide having a sequence of valine-isoleucine-proline-valine-proline-phenylalanine (Val-Ile-Pro-Val-Pro-Phe-Phe) derived from an endogenous protein of Saccharomyces cerevisiae (strain ATCC 204508/S288 c): enolase-related protein 2 (protein accession No. P0CX 11). The theoretical isoelectric point of the small-molecule heptapeptide is 5.49. The molecular weight of the small molecular polypeptide is 818.12 g/mol.
Example 2
The heptapeptide is artificially synthesized by adopting an Fmoc solid-phase synthesis method. According to the composition of amino acid residues of heptapeptides, various amino acids (Fmoc-Phe, Fmoc-Val, Fmoc-Ile and Fmoc-Pro) with Fmoc-protecting groups at amino terminals are used as raw materials, and the carboxyl of the Fmoc-Phe is connected with high molecular resin (Wang resin) through covalent bonds; adding Dimethylformamide (DMF) containing 20% (v/v) piperidine, and reacting for 0.5h to remove the amino protecting group Fmoc-; adding excessive Fmoc-Phe, using Hydroxybenzotriazole (HOBT) as a condensing agent, and reacting for 2h at 30 ℃ to condense carboxyl of the Fmoc-Phe with reactive amino of the Phe on the resin; and repeating the deprotection and condensation reaction, sequentially connecting the rest other amino acids, cracking the heptapeptide from the resin, separating and purifying by a C18 column, and freeze-drying to obtain the ACE inhibitory heptapeptide. The liquid chromatogram (figure 1) analysis shows that the purity of the small molecule polypeptide synthesized by the method is 98.83%. The liquid chromatography-mass spectrometry (fig. 2) confirmed that the sequence of the polypeptide synthesized was valine-isoleucine-proline-valine-proline-phenylalanine (Val-Ile-Pro-Val-Pro-Phe).
Example 3
The small molecular polypeptides are prepared into solutions with the concentration of 1-100 mu g/mL by using borate buffer solution. Respectively taking 100 mu L of small molecule polypeptide solution with different concentrations and 50 mu L of 1.55mmol/L HHL (equuria acyl-histidyl-leucine) solution (solvent is borate buffer solution), preserving the temperature for 5min at 37 ℃, adding 10 mu L of 0.1U/mL ACE solution (solvent is borate buffer solution), uniformly mixing, preserving the temperature for reaction at 37 ℃ for 30min, and adding 80 mu L of 1.0mol/L HCl to terminate the reaction. Meanwhile, 100. mu.L of borate buffer was used instead of the sample solution to prepare a reaction solution as a blank control. The peak area of hippuric acid is detected by RP-HPLC after the reaction solution is filtered by a filter membrane of 0.22 mu m, and the peak area is compared with a standard curve to calculate the amount of the hippuric acid product.
RP-HPLC detection conditions: agilent C18Column (4.6 mm. times.250 mm, 5 μm), mobile phase A deionized water (containing 0.1% (v/v) trifluoroacetic acid TFA), B methanol (containing 0.1% TFA (v/v), A: B ═ 40:60, column temperature 25 ℃, flow rate 0.8mL/min, detection wavelength 228nm, sample size 20 μ L.
semi-Inhibitory Concentration (IC)50) Is the concentration of the sample required when the ACE inhibition reaches 50%. Preparing sample solutions with different concentrations respectively, determining ACE activity inhibition rate, fitting by GraphPad Prism software log (inhibitor) vs. again/Variable slope (four parameters) with the sample concentration as abscissa and the ACE inhibition rate as ordinate, and calculating IC50The value is obtained.
As can be seen from FIG. 3, the heptapeptide of the present invention has inhibitory effect on ACE at different concentrations, and the half inhibitory concentration IC is calculated5016.43. mu.M. The small molecular polypeptide has strong ACE inhibitory activity and can be used for developing blood pressure lowering functional foods and medicines.
Example 4
In the ACE inhibitory activity experiment, different concentration gradients are respectively configured for a substrate HHL: 0.39,0.78,1.55 and 3.10 mM. The inhibition of ACE at 20 and 50. mu.g/mL was determined for the heptapeptide VIPVPFF at different substrate concentrations. The Lineweaver-Burk double reciprocal number graph (FIG. 4) is drawn, and the result shows that the inhibition type of the polypeptide VIPVPFF on ACE belongs to competitive inhibition.
Example 5
Primary human umbilical vein endothelial cells were obtained (purchased)In All cells, product number H-001F-C), 5mL of a complete endothelial cell culture medium (containing 10% fetal bovine serum, growth factor, diabody, purchased from All cells, product number: h-004), inoculating 0.25% gelatin coated T25 cell culture flask at 1.0e5 cells/mL density, placing at 37 deg.C and 5% CO2Culturing in an incubator, changing culture solution every 2 days, and subculturing every 4 days. After 3-5 generation cells were cultured to log phase, they were trypsinized and added to a 96-well plate at a density of 1.0e5 cells/mL, 100. mu.L per well, 6 duplicate wells per group, marginal wells and unused wells filled with equal amounts of sterile PBS. When the cells are cultured until the cells grow to more than 80% of the bottom of the holes, the original culture solution is sucked and discarded, and 100 mu L of serum-free culture solution is used for culturing for 12 hours, so that the cells are in the same period. Then 100. mu.L of heptapeptide VIPVPFF with different concentrations is added into each well, 100. mu.L of serum-free culture solution is added into a control group for continuous culture, and 100. mu.L of common ACE inhibitory drug captopril (the concentration is 10e-5M) is taken as a positive control. At 24h and 48h, 20. mu.L MTT (5mg/mL) was added to each well, the culture was carried out for 4h, the liquid in the wells was aspirated, 150. mu.L DMSO was added thereto, the mixture was gently shaken for 10min, and the absorbance at 570nm was measured with a microplate reader. The cell-free medium was added with the same amount of MTT, and after 4 hours the medium was aspirated off, 150. mu.L of DMSO was added as a zero well.
Cell survival (%) (absorbance of experiment group/absorbance of control group)
As can be seen from FIG. 5, the cell viability of the low dose group (150. mu.g/mL) after the heptapeptide VIPVPFF was added to human umbilical vein endothelial cells and cultured for 24h was not significantly different from that of the control group. The cell viability of the high dose group (300. mu.g/mL) was 91.34 + -2.48% of that of the control group. After 48h of culture, the heptapeptide VIPVPFF low-dose group has no significant difference from the high-dose group and the control group. While the positive control captopril at the concentration of 10e-5M showed a very significant decrease in cell viability for 24h and 48h of cell culture. Compared with the common medicine captopril, the heptapeptide VIPVPFF has smaller influence on the proliferation of human umbilical vein endothelial cells and higher safety.
Example 6
In the course of the present study 778 polypeptides were identified from the yeast zymolyte of example 1 by LC-MS/MS. Several of the polypeptides were synthesized by solid phase synthesis and investigated for their ACE inhibitory activity using the method of example 3, which is shown in figure 6 at a concentration of 100 μ g/mL. The heptapeptide VIPVPFF can be found to have the best ACE inhibitory activity, is remarkably superior to other polypeptides, and has outstanding effects, so that the polypeptide VIPVPFF has unpredictability.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> university of southern China's science
<120> heptapeptide for inhibiting angiotensin converting enzyme and application thereof
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>7
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> heptapeptide amino acid sequence
<400>1
Val Ile Pro Val Pro Phe Phe
1 5
Claims (8)
1. A heptapeptide having ACE inhibitory activity, characterized by:
the amino acid sequence is valine-isoleucine-proline-valine-proline-phenylalanine.
2. Heptapeptides with ACE inhibitory activity according to claim 1, characterized in that:
prepared by solid phase synthesis.
3. Use of the heptapeptide having ACE inhibitory activity according to any one of claims 1 or 2 in a functional food for lowering blood pressure.
4. Use of a heptapeptide having ACE inhibitory activity according to any one of claims 1 or 2 in the preparation of an ACE inhibitor.
5. Use of a heptapeptide having ACE inhibitory activity according to any one of claims 1 or 2 in the manufacture of a medicament for lowering blood pressure.
6. A functional food for reducing blood pressure is characterized in that:
comprising the heptapeptide having ACE inhibitory activity according to any one of claims 1 or 2.
7. An ACE inhibitor characterized by:
comprising the heptapeptide having ACE inhibitory activity according to any one of claims 1 or 2.
8. A blood pressure lowering medicine is characterized in that:
comprising the heptapeptide having ACE inhibitory activity according to any one of claims 1 or 2.
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| CN102286591A (en) * | 2011-09-15 | 2011-12-21 | 华南理工大学 | Preparation method yeast resource active polypeptide |
| CN102311484A (en) * | 2011-09-15 | 2012-01-11 | 华南理工大学 | Hexapeptide for inhabiting angiotensin transferase and preparation method thereof |
| WO2012104462A1 (en) * | 2011-02-04 | 2012-08-09 | Consejo Superior De Investigaciones Científicas (Csic) | Heptapeptides and the use thereof for controlling hypertension |
| CN109485700A (en) * | 2018-12-05 | 2019-03-19 | 广西大学 | A kind of angiotensin converting enzyme ace inhibitory peptide and preparation method thereof |
| CN110467653A (en) * | 2019-09-12 | 2019-11-19 | 浙江省农业科学院 | A kind of active polypeptide of inhibition Angiotensin-Converting and its application |
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| WO2012104462A1 (en) * | 2011-02-04 | 2012-08-09 | Consejo Superior De Investigaciones Científicas (Csic) | Heptapeptides and the use thereof for controlling hypertension |
| CN102286591A (en) * | 2011-09-15 | 2011-12-21 | 华南理工大学 | Preparation method yeast resource active polypeptide |
| CN102311484A (en) * | 2011-09-15 | 2012-01-11 | 华南理工大学 | Hexapeptide for inhabiting angiotensin transferase and preparation method thereof |
| CN109485700A (en) * | 2018-12-05 | 2019-03-19 | 广西大学 | A kind of angiotensin converting enzyme ace inhibitory peptide and preparation method thereof |
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