CN116041428B - Two ACE (angiotensin converting enzyme) inhibitory peptides as well as preparation method and application thereof - Google Patents
Two ACE (angiotensin converting enzyme) inhibitory peptides as well as preparation method and application thereof Download PDFInfo
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Classifications
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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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- 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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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Heart & Thoracic Surgery (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Mycology (AREA)
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Abstract
The invention discloses two ACE inhibitory peptides and a preparation method and application thereof, wherein the ACE inhibitory peptides comprise one or a combination of two polypeptides as follows: 1) A polypeptide with an amino acid sequence LVPYRP (SEQ ID No. 1), 2) a polypeptide with an amino acid sequence WYWPQ (SEQ ID No. 2). The invention also provides a preparation method and application of the ACE inhibitory peptide, and the two ACE inhibitory peptides have good intestinal absorbability and ACE inhibitory effect of human bodies, are nontoxic and have good market prospect in the pharmaceutical industry.
Description
Technical Field
The invention belongs to the technical field of biology, relates to ACE inhibitory peptides and a preparation method and application thereof, and particularly relates to two polypeptides with ACE inhibitory activity, which are derived from millet proteins, and application thereof.
Background
Hypertension is a common chronic disease and is mainly characterized by increased arterial blood pressure (systolic pressure and/or diastolic pressure) of the systemic circulation, which increases the risk of injury to heart, brain, kidney and other visceral functions or organic matters, such as coronary heart disease, cerebrovascular disease, renal failure and the like. Research has shown that hypertension is often accompanied by the occurrence of diseases such as diabetes, atherosclerosis and the like.
The most common drugs currently used clinically to treat hypertension are angiotensin-converting enzyme (ACE) inhibitors, which inhibit the conversion of angiotensin-converting enzyme I to angiotensin-converting enzyme II in the renin-angiotensin system, bradykinin inactivation in the kinin-kininogenase system, and increase in blood pressure in both pathways. At present, the traditional ACE inhibitor is an artificial synthetic drug, such as enalapril, captopril and the like, and the drug has remarkable effect, but is often accompanied with side effects such as dizziness, cough, nausea and the like. At present, food-derived polypeptides become a new research object in the field of blood pressure reduction, and in general, most of the peptides have smaller multi-phase molecular mass and are easier to digest and absorb proteins in a human body. The small peptides not only can provide nutrition required by the growth and development of human bodies, but also can regulate the physiological functions of the human bodies, and play roles in preventing and even treating diseases. Therefore, the polypeptide with ACE inhibitory activity obtained from the food protein has better development prospect as a safe substitute for preventing and treating hypertension.
Disclosure of Invention
Aiming at the problems and the defects of the existing antihypertensive drugs, the invention provides a functional polypeptide with ACE inhibitory activity, and a preparation method and application thereof.
In one aspect, embodiments of the present invention provide ACE inhibiting peptides comprising one or a combination of two polypeptides as follows:
1) A polypeptide with an amino acid sequence LVPYRP (SEQ ID No. 1),
2) A polypeptide having the amino acid sequence WYWPQ (seq id No. 2).
The ACE inhibitory peptides of the embodiment of the invention are LVPYRP (Leu-Val-Pro-Tyr-Arg-Pro, leucine-valine-proline-tyrosine-arginine-proline, SEQ ID No. 1) and WYWPQ (Trp-Tyr-Trp-Pro-Gln, tryptophan-tyrosine-tryptophan-proline-glutamine, SEQ ID No. 2) respectively, and the two polypeptides have good intestinal tract absorbability and ACE inhibitory effect of human bodies, are nontoxic, have clear action mechanism and clear targets, meet the requirements of pharmaceutical preparation development, and have good market prospects in the pharmaceutical industry.
In another aspect, embodiments of the present invention provide a composition comprising an ACE inhibiting peptide as described above and a pharmaceutically acceptable carrier.
In another aspect, the present invention further provides an application of the ACE inhibiting peptide or the composition in preparation of an ACE inhibitor.
In another aspect, the invention also provides application of the ACE inhibitory peptide or the composition in preparation of products for treating and/or preventing hypertension.
In some embodiments, the product comprises a medicament.
The embodiment of the invention also provides a millet protein hydrolysate rich in ACE inhibitory peptide, which comprises two peptide fragments with ACE inhibitory activity, wherein the amino acid sequences of the two peptide fragments with ACE inhibitory activity are LVPYRP (SEQ ID No. 1) and WYWPQ (SEQ ID No. 2).
According to the invention, the millet protein is subjected to enzymolysis by alkaline protease to obtain an enzymolysis liquid, the enzymolysis liquid is subjected to ultrafiltration, preparative liquid chromatography separation and liquid chromatography-tandem mass spectrometry analysis to obtain a polypeptide sequence, and finally, the Fmoc solid-phase synthesis method is used for preparing the polypeptide, and the ACE inhibition activity of the polypeptide is verified.
The embodiment of the invention also provides a preparation method of the ACE inhibitory peptide, which comprises the following steps:
(1) Hydrolyzing millet protein by adopting alkaline protease to obtain millet protein enzymolysis liquid;
(2) Carrying out ultrafiltration treatment on millet protein enzymolysis liquid, taking components with molecular weight less than 3 kDa, and freeze-drying to obtain ACE inhibition crude peptide;
(3) Further separating ACE inhibition crude peptide by preparative liquid chromatography, and freeze-drying the component with optimal ACE inhibition activity by taking ACE inhibition activity as an evaluation index;
(4) The amino acid sequence of the component with the optimal ACE inhibitory activity is determined by liquid chromatography-tandem mass spectrometry to determine the amino acid compositions thereof to be LVPYRP (SEQ ID No. 1) and WYWPQ (SEQ ID No. 2), and the Fmoc solid-phase synthesis method is adopted to prepare the polypeptide.
In some embodiments, the above method further comprises: further functional evaluation, namely, carrying out ACE activity inhibition experiments on the target peptide segment synthesized artificially to evaluate the actual ACE inhibition effect, and finally evaluating toxicity, isoelectric point, total average hydrophilicity and human intestinal tract absorbability based on computer software.
In some embodiments, in step (1), the enzymatic hydrolysis is performed at a pH of 8.0 and at a temperature of 60℃for a time of 4 h.
In some embodiments, the ultrafiltration treatment refers to obtaining three components having molecular weights MW <3 kDa, 3-10 kDa, and >10 kDa, respectively, by ultrafiltration membranes having molecular weights of 3 kDa and 10 kDa.
In some embodiments, the millet protein is obtained from the enzymatic hydrolysis of defatted millet flour using a three-enzyme combination of an alpha-amylase, a saccharifying enzyme, and a complex cellulase.
The invention has the following advantages and beneficial effects:
The two functional polypeptides (LVPYRP (SEQ ID No. 1) and WYWPQ (SEQ ID No. 2)) discovered from millet protein are purely natural, nontoxic and harmless plant source substances, and have remarkable ACE inhibition effects. Human body regulates blood pressure through renin-angiotensin system and kinin-kinin producing enzyme system, ACE plays an important role in regulating balance of two systems, and is often studied extensively as a target for controlling blood pressure level. LVPYRP (SEQ ID No. 1) and WYWPQ (SEQ ID No. 2) are effective in lowering blood pressure by inhibiting ACE activity, and this is also confirmed by in vitro enzyme activity inhibition experiments. Therefore, LVPYRP (SEQ ID No. 1) and WYWPQ (SEQ ID No. 2) of the invention are used as antihypertensive components, have good ACE inhibition effect, are nontoxic, have clear action mechanism and definite target points, meet the requirements of pharmaceutical preparation development, and have good market prospects in the pharmaceutical industry.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings, in which:
FIG. 1 shows the inhibition of ACE activity by different ultrafiltration fractions.
FIG. 2 is a liquid chromatography separation of active peptides.
Figure 3 shows the inhibitory effect of the liquid phase components on ACE activity.
Fig. 4 is a mass spectrum of ACE inhibitory peptide (amino acid sequence LVPYRP (seq id No. 1)).
Fig. 5 is a mass spectrum of ACE inhibitory peptide (amino acid sequence WYWPQ (seq id No. 2)).
Fig. 6 shows the ACE inhibitory effect of two ACE inhibitory peptides.
Detailed Description
The following detailed description of embodiments of the invention is exemplary and intended to be illustrative of the invention and not to be construed as limiting the invention.
The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
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 terms "comprising," "including," and "comprising" are open-ended, meaning the terms including the elements of the invention, but not excluding other elements.
The terms "peptide", "polypeptide", "peptide fragment" refer to a molecular chain of amino acid residues, which may be modified at each of its amino acid residues, if desired, for example by mannosylation (manosylation), glycosylation, amidation (e.g., C-terminal amide), carboxylation or phosphorylation. The peptide may be obtained synthetically, via genetic engineering methods, expressed in host cells, or via any other suitable means. Methods for producing peptides are well known in the art.
The term "prevention" refers to a reduction in the risk of acquiring a disease or disorder, i.e.: at least one clinical symptom of the disease is stopped from developing in a subject who may be facing or predisposed to facing the disease, but has not yet experienced or exhibited symptoms of the disease.
The term "treating" refers to ameliorating a disease or disorder, i.e.: slowing or preventing or alleviating the progression of the disease or at least one clinical symptom thereof.
The term "pharmaceutically, food or nutraceutical acceptable carrier" refers to any formulation or carrier medium capable of delivering an effective amount of the active substance (ACE inhibiting peptide) of the present invention, without interfering with the biological activity of the active substance and without toxic or side effects to the host or subject. Including but not limited to: one or more of diluents, binders, disintegrants, lubricants, wetting agents, thickeners, preservatives, antioxidants, pH adjusters, solvents, cosolvents, surfactants, opacifiers, etc.
According to the embodiment of the invention, ACE inhibitory peptide in millet protein hydrolysate is screened out through ultrafiltration, liquid chromatography, mass spectrometry sequencing and other technologies, and functional verification is performed, so that technical support is provided for developing a safe substitute of a antihypertensive drug.
In one aspect, embodiments of the present invention provide ACE inhibiting peptides comprising one or a combination of two polypeptides:
1) A polypeptide with an amino acid sequence LVPYRP (SEQ ID No. 1),
2) A polypeptide having the amino acid sequence WYWPQ (seq id No. 2).
The ACE inhibitory peptides of the embodiment of the invention are LVPYRP (Leu-Val-Pro-Tyr-Arg-Pro, leucine-valine-proline-tyrosine-arginine-proline, SEQ ID No. 1) and WYWPQ (Trp-Tyr-Trp-Pro-Gln, tryptophan-tyrosine-tryptophan-proline-glutamine, SEQ ID No. 2) respectively, and the two polypeptides have good intestinal tract absorbability and ACE inhibitory effect of human bodies, are nontoxic, have clear action mechanism and clear targets, meet the requirements of pharmaceutical preparation development, and have good market prospects in the pharmaceutical industry.
In another aspect, the present invention provides a composition comprising the ACE inhibiting peptide and a pharmaceutically acceptable carrier.
In another aspect, the present invention further provides an application of the ACE inhibiting peptide or the composition in preparation of an ACE inhibitor.
In another aspect, the invention also provides application of the ACE inhibitory peptide or the composition in preparation of products for treating and/or preventing hypertension.
In some embodiments, the product comprises a drug.
The embodiment of the invention also provides a millet protein hydrolysate rich in ACE inhibitory peptide, which comprises two peptide fragments with ACE inhibitory activity, wherein the amino acid sequences of the two peptide fragments with ACE inhibitory activity are LVPYRP (SEQ ID No. 1) and WYWPQ (SEQ ID No. 2).
According to the invention, the millet protein is subjected to enzymolysis by alkaline protease to obtain an enzymolysis liquid, the enzymolysis liquid is subjected to ultrafiltration, preparative liquid chromatography separation and liquid chromatography-tandem mass spectrometry analysis to obtain a polypeptide sequence, and finally, the Fmoc solid-phase synthesis method is used for preparing the polypeptide, and the ACE inhibition activity of the polypeptide is verified.
In another aspect, the embodiment of the invention also provides a preparation method of ACE inhibitory peptide, which comprises the following steps:
(1) Hydrolyzing millet protein by adopting alkaline protease to obtain millet protein enzymolysis liquid;
(2) Carrying out ultrafiltration treatment on millet protein enzymolysis liquid, taking components with molecular weight less than 3 kDa, and freeze-drying to obtain ACE inhibition crude peptide;
(3) Further separating ACE inhibition crude peptide by preparative liquid chromatography, and freeze-drying the component with optimal ACE inhibition activity by taking ACE inhibition activity as an evaluation index;
(4) The amino acid sequence of the component with the optimal ACE inhibitory activity is determined by liquid chromatography-tandem mass spectrometry to determine the amino acid compositions thereof to be LVPYRP (SEQ ID No. 1) and WYWPQ (SEQ ID No. 2), and the Fmoc solid-phase synthesis method is adopted to prepare the polypeptide.
In some embodiments, the above method further comprises a further functional evaluation, namely, performing an ACE activity inhibition experiment on the artificially synthesized target peptide fragment to evaluate the actual ACE inhibition effect, and finally evaluating the toxicity, isoelectric point, total average hydrophilicity and human intestinal tract absorbability based on computer software.
In some embodiments, in step (1), the enzymatic hydrolysis is performed at a pH of 8.0 and at a temperature of 60℃for a time of 4 h.
In some embodiments, in step (1), the alkaline protease is added at a ratio of 4000U/g.
In some embodiments, ultrafiltration treatment refers to obtaining three components having molecular weights MW <3 kDa, 3-10 kDa, and >10 kDa, respectively, by ultrafiltration membranes having molecular weights of 3 kDa and 10 kDa.
In some embodiments, in step (3), the liquid chromatography column is Waters XBridge Prep C (250 mm ×19 mm, 5 μm) and the column temperature is 30 ℃, eluting with a mixed gradient, a:0.1% TFA water; b:0.1% TFA acetonitrile. The gradient elution procedure was as follows: 0-5 min,10% B, 5-15 min,10-35% B;15-40 min,35-70% B;40-45 min,10% B. The loading was 2mL, the concentration was 2 mg/mL, and the absorbance peak was measured at 218: 218 nm.
In some embodiments, the millet protein is obtained by enzymatic hydrolysis of defatted millet flour using a three-enzyme combination of an alpha-amylase, a saccharifying enzyme, and a complex cellulase, in a specific example, the ratio of alpha-amylase, saccharifying enzyme, and complex cellulase is 2:2:1.
Example 1 preparation of millet protein
Sieving semen Setariae powder with 60 mesh sieve, mixing semen Setariae powder and n-hexane at a ratio of 1:5 (w/v), degreasing in water bath shaker at 37deg.C for 4 h, recovering n-hexane, and naturally air-drying semen Setariae powder in fume hood for 24: 24 h. The millet protein is obtained by adopting an alpha-amylase, saccharifying enzyme and compound cellulase to carry out enzymolysis on defatted millet powder by a three-enzyme compound method. The extraction process is pH 4.7, the enzymolysis temperature is 48 ℃, the enzyme adding amount is 2.2% of the mass of the millet powder (the mass ratio of alpha-amylase to saccharifying enzyme to compound cellulase is 2:2:1), and the enzymolysis time is 10 h. The enzyme was then deactivated and centrifuged at 7000 rpm for 20: 20 min, the supernatant was discarded, the pellet was washed with water several times and the pH was adjusted to neutral. Finally, freeze-dried and stored at-20 ℃.
Example 2 preparation of millet proteolytic liquid
Millet protein and distilled water were mixed in a ratio of 1:10 (w/v), and then alkaline protease was added to the solution in a ratio of 4000U/g, the enzymatic hydrolysis pH was 8.0, the enzymatic hydrolysis temperature was 60℃and the enzymatic hydrolysis time was 4 h. After the enzymolysis is finished, boiling water is heated to inactivate enzyme 5min, and the enzymolysis liquid is centrifugated to obtain supernatant liquid, freeze-dried and stored at the temperature of minus 20 ℃ for standby.
EXAMPLE 3 preparation of ACE inhibitory crude peptides
For purification of millet ACE inhibitory peptides, the enzymatic hydrolysate was formulated at a concentration of 10 mg/mL as a primary solution (MPH). MPH was sequentially passed through ultrafiltration membranes of molecular weights 3 kDa and 10 kDa to obtain three fractions of molecular weights MW <3 kDa, 3-10 kDa and >10 kDa, respectively, which were lyophilized and stored at-20 ℃ for subsequent ACE inhibition activity evaluation. The effect of the three components (> 10 kDa, 3-10 kDa and <3 kDa) on ACE activity at a concentration of 1 mg/mL was measured and the results indicated that the <3 kDa fraction showed the best inhibitory effect on ACE activity (see figure 1). The ACE activity inhibition experiment comprises the following specific processes:
Preparation of group Ma Niaoxian aminoacyl leucine (HHL) solution: accurately weighing 0.107 g HHL and 0.877 g NaCl, weighing 5 ml sodium borate buffer (pH 8.3), and fixing the three with ultrapure water in a 50 ml volumetric flask for standby.
150. Mu.L of the sample was thoroughly mixed with 100. Mu.L (100 mU/mL) of ACE at 37℃for 10: 10 min, then 500. Mu.L of HHT solution was added, 60: 60 min reacted at 37℃and then 750. Mu.L of HCl (1 mol/L), 1500. Mu.L of pyridine, 750. Mu.L of benzenesulfonyl chloride were added successively. The solution was vortexed 1 min and immediately cooled in an ice bath and absorbance was measured at wavelength 410 nm. Water was used instead of the sample as a control and water was used instead of the HHL solution as a blank.
ACE inhibition was calculated according to formula (1):
ACE inhibition rate (%) = X 100 type (1)
In the formula (1), A C is absorbance of a control group; a S -absorbance of sample; a B -absorbance of blank.
EXAMPLE 4 screening of ACE inhibitory peptides
Separating alkaline protease hydrolysate with molecular weight less than 3 kDa by preparative liquid chromatography, wherein the chromatographic column is Waters XBridge Prep C (250 mm ×19 mm, 5 μm) and the column temperature is 30deg.C, eluting with mixed gradient, A:0.1% TFA water; b:0.1% TFA acetonitrile. The gradient elution procedure was as follows: 0-5 min,10% B, 5-15 min,10-35% B;15-40 min,35-70% B;40-45 min,10% B. The loading was 2mL at a concentration of2 mg/mL and the absorbance peak was measured at 218: 218 nm and collected for use (see FIG. 2). The effect of each absorption peak (fractions 1-7) on ACE activity at a concentration of 1 mg/mL was then measured and indicated that fraction 4 had the best inhibitory effect on ACE activity (see FIG. 3), lyophilized and stored at-20℃for use.
EXAMPLE 5 Synthesis of ACE inhibitory peptides
According to the results of the ACE inhibitory activity analysis of step (4), the amino acid sequence of component 4 was analyzed using a Q Exactive ™ mass spectrometer of tandem EASY-nanoLC, and the sample was analyzed via LC-MS/MS equipped with an online nano-spray ion source. The whole set of system is a Q Exactive ™ mass spectrometer (Thermo FISHER SCIENTIFIC, MA, USA) of a tandem EASY-nanoLC. A total of 3. Mu.L of sample was applied (analytical column: ACCLAIM PEPMAP C, 75. Mu. m x 25, cm), the sample was separated with a 60min gradient, the column flow was controlled at 400 nL/min, the column temperature was 40 ℃, the electrospray voltage was 2 kV, the gradient was started from 0% phase B, the gradient was increased to 60% with a nonlinear gradient at 46 min, the increase was 100% in 4 min, and the duration was 10 min.
The mass spectrometer operates in a data dependent acquisition mode, automatically switching between MS and MS/MS acquisition. The mass spectral parameters were set as follows: (1) MS: scan range (m/z): 200-1800, resolution: 70,000;AGC target:3e6, a maximum injection time of 60MS, and (2) HCD-MS/MS, a resolution of 17,500;AGC target:5e4, maximum injection time 80ms, collision energy 27, dynamic exclusion time 20s. The amino acid compositions of the peptides are determined to be LVPYRP (SEQ ID No. 1) and WYWPQ (SEQ ID No. 2), the two polypeptides are prepared by Fmoc solid-phase synthesis method, and the purity of each peptide is determined to be more than 95% through HPLC chromatography and mass spectrometry analysis, and mass spectrograms of the two polypeptides are shown in FIG. 4 and FIG. 5.
Example 6 functional evaluation
The ACE activity inhibition experiments show that IC 50 of LVPYRP (SEQ ID No. 1) and WYWPQ (SEQ ID No. 2) are 0.2365 and 0.4023 mg/ml respectively (see FIG. 6).
Computer software was used to perform functional predictions for LVPYRP (seq id No. 1) and WYWPQ (seq id No. 2), wherein:
toxicity and steric hindrance by ToxinPred (https:// webs. Iiitd. Edu. In/raghava/toxinpred/index. Html), total average hydrophilicity by ExPasy (https:// web. ExPasy org/protparam /).
Human intestinal absorption is achieved by admetSAR (http:// lmmd.ecl.edu.cn/admetsar 1/home /),
Isoelectric points were assessed by Pepdraw (http:// www.tulane.edu/-biochem/WW/PepDraw /).
As shown in Table 1, both polypeptides were non-toxic and had good intestinal absorption in humans. LVPYRP (SEQ ID No. 1) has an isoelectric point of 10.35 and is alkaline; WYWPQ (SEQ ID No. 2) has an isoelectric point of 5.56 and is acidic. The total average hydrophilicity can be used to characterize the hydrophilicity and hydrophobicity of a protein, where a larger positive value indicates a stronger hydrophobicity and a larger negative value indicates a stronger hydrophilicity, so LVPYRP (SEQ ID No. 1) and WYWPQ (SEQ ID No. 2) have a certain hydrophilicity.
TABLE 1 ACE functional prediction of inhibitory peptides
Note that: +/-represents the good/poor intestinal absorption of the polypeptide, respectively
For purposes of this disclosure, the terms "one embodiment," "some embodiments," "example," "a particular example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (6)
- ACE inhibiting peptide, characterized in that it is a combination of one or two polypeptides as follows:1) The amino acid sequence is polypeptide shown as SEQ ID No.1,2) The amino acid sequence is polypeptide shown as SEQ ID No. 2.
- 2. A composition comprising the ACE inhibiting peptide of claim 1 and a pharmaceutically acceptable carrier.
- 3. Use of the ACE inhibiting peptide of claim 1 or the composition of claim 2 in the preparation of an ACE inhibitor.
- 4. Use of an ACE inhibiting peptide as claimed in claim 1 or a composition as claimed in claim 2 for the preparation of a product for the treatment and/or prophylaxis of hypertension.
- 5. The use according to claim 4, wherein the product comprises a medicament.
- 6. An ACE inhibitory peptide-enriched millet protein hydrolysate, characterized in that: comprises two peptide fragments with ACE inhibitory activity, and the amino acid sequences of the two peptide fragments with ACE inhibitory activity are SEQ ID No.1 and SEQ ID No.2.
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| JP2012067041A (en) * | 2010-09-24 | 2012-04-05 | Oriza Yuka Kk | Ace activity inhibitor |
| CN104450839A (en) * | 2014-11-05 | 2015-03-25 | 哈尔滨商业大学 | Preparation method of rice bran protein peptide with ACE inhibitory activity |
| CN104945502A (en) * | 2015-06-30 | 2015-09-30 | 石狮海星食品有限公司 | ACE (angiotensin converting enzyme) inhibitory pentapeptide |
| TW201623622A (en) * | 2014-12-31 | 2016-07-01 | 嘉藥學校財團法人嘉南藥理大學 | Method of preparing fermentation crude extracts for inhibiting activity of angiotensin I -converting enzyme |
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| JP2012067041A (en) * | 2010-09-24 | 2012-04-05 | Oriza Yuka Kk | Ace activity inhibitor |
| CN104450839A (en) * | 2014-11-05 | 2015-03-25 | 哈尔滨商业大学 | Preparation method of rice bran protein peptide with ACE inhibitory activity |
| TW201623622A (en) * | 2014-12-31 | 2016-07-01 | 嘉藥學校財團法人嘉南藥理大學 | Method of preparing fermentation crude extracts for inhibiting activity of angiotensin I -converting enzyme |
| CN104945502A (en) * | 2015-06-30 | 2015-09-30 | 石狮海星食品有限公司 | ACE (angiotensin converting enzyme) inhibitory pentapeptide |
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