CN106554390A - A kind of polypeptide and its application in preparation ACE inhibitor or Altace Ramipril - Google Patents
A kind of polypeptide and its application in preparation ACE inhibitor or Altace Ramipril Download PDFInfo
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- CN106554390A CN106554390A CN201510622913.1A CN201510622913A CN106554390A CN 106554390 A CN106554390 A CN 106554390A CN 201510622913 A CN201510622913 A CN 201510622913A CN 106554390 A CN106554390 A CN 106554390A
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Abstract
The present invention relates to suppress Angiotensin-Converting (angiotensin-converting enzyme, ACE a kind of) polypeptide compound QEPVLGPVRGPFPIIV of activity and blood pressure lowering, its aminoacid sequence are Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile- Ile-Val.Polypeptide QEPVLGPVRGPFPIIV has ACE inhibitory activity and hypotensive activity, has a good application prospect as the health product and lead compound of hypertension, heart disease and cardiovascular diseasess.
Description
Technical field
The present invention relates to polypeptide QEPVLGPVRGPFPIIV is preparing suppression angiotensin
Converting Enzyme (ACE) and Altace Ramipril and the application in health product.
Background technology
Hypertension is a kind of common cardiovascular disease, and sickness rate is high, be the initiation heart, brain, kidney
With the various complication such as blood vessel and cause apoplexy, promote atherosclerosiss, one of coronary heart disease
The significant risk factor.The average attack rate of countries in the world be 10%-20%, China hyperpietic
More than 1.6 hundred million populations, it is the highly important problem of current social to treat and prevent hypertension.
Peptide is the important Altace Ramipril of a class, and the action target spot of peptides blood pressure lowering is to suppress blood
Activity (the text of angiotensin invertase (angiotensin-converting enzyme, ACE)
Offer 1：Vanessa Vermeirssen, John Van Camp, Willy Verstraete,
British Journal of Nutrition 2004,92：357-366).Vasotonia
It is with strong contraction blood that plain invertase can not be had active decapeptide angiotensin I converting
The octapeptide Angiotensin II of pipe effect, so that blood pressure is raised, therefore suppresses ACE active
Can effective control hypertension.Polypeptide is the important ACE inhibitor of a class, native protein
Peptides are the main source (documents 2 of ACE inhibitor peptide：Lieselot Vercruysse,
John Van Camp,Guy Smagghe,J.Agric.Food Chem 2005,53:
8106-8115).Angiotensin converting enzyme is important for body blood pressure and cardiovascular function are played
Adjustment effect, therefore suppress ACE activity medicine in diseases such as cardiovascular, heart failure
Play a significant role in treatment.The ACE inhibitor of polypeptide will not be drawn while blood pressure lowering
Play the side effect such as the dry cough of common antihypertensive drugs.
The content of the invention
It is an object of the invention to provide polypeptide QEPVLGPVRGPFPIIV is suppressing ACE to live
Application and rapid screening method in property and blood pressure lowering；Polypeptide QEPVLGPVRGPFPIIV
With ACE inhibitory activity and hypotensive activity, as hypertension, heart disease and cardiovascular diseasess
Health product and lead compound have a good application prospect.
For achieving the above object, the present invention with the polypeptide QEPVLGPVRGPFPIIV is
Suppress the active ingredient of ACE activity and blood pressure lowering.
Which has sequence table SEQ ID NO：Aminoacid sequence in 1；Polypeptide
QEPVLGPVRGPFPIIV is the activity of ACE inhibitor and Altace Ramipril and health product
Composition, wherein pharmaceutically acceptable carrier or adjuvant can be added.
With the polypeptide QEPVLGPVRGPFPIIV for suppressing ACE activity and hypotensive activity,
Aminoacid sequence is Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-
Pro-Ile-Ile-Val, is single-stranded linear structure, and white powder is soluble in water, and molecular weight is
1717Da；There is to ACE activity good inhibiting effect, IC50 is 160.48 μM.
Polypeptide QEPVLGPVRGPFPIIV possesses the feature required by ACE inhibitor：
1.ACE inhibitory activity peptides depend primarily on C-terminal aminoacid, and C-terminal is aromatic amine
Base acid (Trp, Phe, Tyr), or hydrophobic amino acid, and N-terminal is the peptide fragment of hydrophobic amino acid
With stronger ACE inhibitory activity.The C-terminal six of polypeptide QEPVLGPVRGPFPIIV
Peptide is above-mentioned aminoacid, respectively Pro (hydrophobic), Phe (aromatic series, hydrophobic), Pro
(hydrophobic), Ile (hydrophobic), Ile (hydrophobic), Val (hydrophobic), therefore part meets ACE
Feature required by inhibitor, with preferable ACE inhibitory activity.
2. the hydrophobic amino acid content of peptide is the major reason for affecting its inhibitory activity, is suppressed
The high peptide of activity all contains more hydrophobic amino acid.Polypeptide QEPVLGPVRGPFPIIV
Hydrophobic amino acid have respectively Pro, Val, Leu, Pro, Val, Pro, Phe, Pro, Ile,
Ile, Val, containing more hydrophobic amino acid.
The present invention compared with prior art, has the advantages that：
The present invention obtains and determines the structure of reactive compound, compound tool first from Yoghourt
There is the activity for preferably suppressing ACE, therefore as cardiovascular disease medicines such as treatment hypertension
Lead compound there is good potentiality and application prospect.
Specific embodiment
The preparation of 1 polypeptide QEPVLGPVRGPFPIIV of embodiment
The method combined with Shotgun proteomic techniques using LC-MS/MS.With cattle
Milk Yoghourt be raw material, Jing enzymolysis proteins, centrifugation, ultrafiltration and LC-MS/MS analysis, with reference to
Structure activity relationship feature, screens the peptide fragment inhibited to ACE.
Its concrete grammar is as follows：Yoghurt example and 35mM NaCl solutions are according to 1：1 volume
Than mixing, it is 2.0 with 1M salt acid for adjusting pH, determines the protein content in Yoghourt, add
Pepsin, addition are pepsin：Albumen quality ratio=1 in yoghurt example:100, in 37 DEG C
Enzymolysis 1h.1M NaOH adjust pH to 7.0 and terminate reaction.Enzymolysis solution liquid enzymolysis solution in 4 DEG C,
30 minutes (10～60 minutes) are centrifuged under 8000g rotating speeds (3000～10000g) anti-
Liquid is answered, supernatant molecular cut off is 8000 ultrafilter membrane (3000～10000) ultrafiltration,
C18-SPE posts desalination after filtrate.By enzymolysis sample lyophilizing after desalination, mass concentration 0.1% is dissolved in
In formic acid water, LTQ Orbitrap XL (ion trap cyclotron resonance combines mass spectrograph) are carried out to sample
Mass spectral analyses.
Peptide sequence searches storehouse：Sample is carried out into mass spectral analyses, ion source with LTQ Orbitrap XL
For ESI, the data for obtaining in bovine.fasta storehouses are entered line retrieval, are carried out with reference to structure activity relationship
Screening, obtains polypeptide of the sequence for QEPVLGPVRGPFPIIV.
The ACE inhibitory activity detection of 2 polypeptide QEPVLGPVRGPFPIIV of embodiment
Principle
The simulation of ACE catalytic decomposition angiotensin Is under conditions of 37 DEG C, PH8.3
Substrate Hippuryl-L-Histidyl-L-Leucine (HHL) produces hippuric acid, and the material exists
There is feature ultraviolet absorption peak at 228nm.When ACE mortifiers are added, ACE is to HHL
Catalytic action be suppressed, the growing amount of hippuric acid can be reduced.Inhibitor is added by determining
Hippuric acid ultraviolet absorption value in front and back can calculate the size of inhibitory activity.
Reaction system
Buffer is 0.05M, PH8.3 borate buffer solutions；Substrate is
Hippuryl-L-Histidyl-L-Leucine (HHL), MW 429.47, are matched somebody with somebody with above-mentioned buffer
Into 5mM；ACE (angiotensin-converting enzyme) is made into above-mentioned buffer
0.1U/ml。
ODA(matched group is light absorption value when there is no inhibitor but there is enzyme)：50μl
Buffer+50ulHHL+50 μ l buffer is subsequently adding 50 μ in 37 DEG C of water-bath 5min
LACE, 37 DEG C of water-bath 30min, 200 μ l of addition, the HCl terminating reactions of 1M, then plus
Enter 1ml ethyl acetate extraction product hippuric acids, vibrate 15S, 3500r/min centrifugation 5min,
0.8ml supernatants are taken, 90 DEG C of drying with water bath 15min are dissolved in 0.8ml distilled water, 228nm again
Place's detection light absorption value is ODA。
ODB(sample sets are light absorption value when there is inhibitor and enzyme):50 μ l samples+50
μ lHHL+50 μ l buffer is subsequently adding 50 μ lACE in 37 DEG C of water-bath 5min, 37 DEG C
Water-bath 30min, adds 200 μ l, the HCl terminating reactions of 1M to add 1ml acetic acid
Ethyl ester extraction product hippuric acid, vibrates 15S, 3500r/min centrifugation 5min, takes on 0.8ml
Clearly, 90 DEG C of drying with water bath 15min, are dissolved in 0.8ml distilled water again, and detection at 228nm is inhaled
Light value is ODB。
The polypeptide QEPVLGPVRGPFPIIV obtained using embodiment 1 is carried out as sample
Operation.
ODC(blank group is light absorption value when there is no inhibitor and enzyme)：50 μ l buffer
+ 50 μ lHHL+50 μ l buffer are subsequently adding 50 μ l bufferings in 37 DEG C of water-bath 5min
Liquid, 37 DEG C of water-bath 30min add 200 μ l, the HCl terminating reactions of 1M to add
1ml ethyl acetate extraction product hippuric acids, vibrate 15S, 3500r/min centrifugation 5min, take
0.8ml supernatants, 90 DEG C of drying with water bath 15min are dissolved in 0.8ml distilled water, 228nm again
Place's detection light absorption value is ODC。
ACE suppression ratio (%)=(ODA-ODB)/(ODA-ODC) × 100%
Respectively with different concentration, as stated above to polypeptide QEPVLGPVRGPFPIIV
Carry out ACE inhibitory activity detection.As a result it is as follows：
Polypeptide QEPVLGPVRGPFPIIV has ACE inhibitory activity and hypotensive activity, as
The health product and lead compound of hypertension, heart disease and cardiovascular diseasess have good answering
Use prospect.
Claims (4)
1. a kind of polypeptide, it is characterised in that：The polypeptide is QEPVLGPVRGPFPIIV,
With sequence table SEQ ID NO：Aminoacid sequence in 1；The aminoacid sequence of the polypeptide is concrete
For Gln-Glu-Pro-Val-Leu-Gly-Pro-Val-Arg-Gly-Pro-Phe-Pro-Ile- Ile-Val.
2. described in a kind of claim 1 in ACE inhibitor or Altace Ramipril is prepared should
With.
3. according to the application described in claim 2, it is characterised in that：The ACE inhibitor
And/or Altace Ramipril is with polypeptide QEPVLGPVRGPFPIIV as active ingredient.
4. according to the application described in claim 2, it is characterised in that：The ACE inhibitor
And/or pharmaceutically acceptable carrier or adjuvant in Altace Ramipril, can be added.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510622913.1A CN106554390B (en) | 2015-09-25 | 2015-09-25 | Polypeptide and application thereof in preparing ACE inhibitor or antihypertensive drug |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510622913.1A CN106554390B (en) | 2015-09-25 | 2015-09-25 | Polypeptide and application thereof in preparing ACE inhibitor or antihypertensive drug |
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| Publication Number | Publication Date |
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| CN106554390A true CN106554390A (en) | 2017-04-05 |
| CN106554390B CN106554390B (en) | 2020-01-14 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112694429A (en) * | 2020-12-29 | 2021-04-23 | 江苏医药职业学院 | Polypeptide and application thereof in preparing ACE inhibitor or blood pressure lowering product |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050281914A1 (en) * | 2003-06-20 | 2005-12-22 | Steele James L | Methods and compositions involving endopeptidases PepO2 and PepO3 |
| CN104558110A (en) * | 2013-10-21 | 2015-04-29 | 中国科学院大连化学物理研究所 | Polypeptide with ACE inhibitory activity and application thereof in antihypertensive drug |
-
2015
- 2015-09-25 CN CN201510622913.1A patent/CN106554390B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050281914A1 (en) * | 2003-06-20 | 2005-12-22 | Steele James L | Methods and compositions involving endopeptidases PepO2 and PepO3 |
| US7741438B2 (en) * | 2003-06-20 | 2010-06-22 | Wisconsin Alumni Research Foundation | Methods and compositions involving endopeptidases PepO2 and PepO3 |
| CN104558110A (en) * | 2013-10-21 | 2015-04-29 | 中国科学院大连化学物理研究所 | Polypeptide with ACE inhibitory activity and application thereof in antihypertensive drug |
Non-Patent Citations (3)
| Title |
|---|
| L. ONG, N.P. SHAH等: "Release and identification of angiotensin-converting enzyme-inhibitory peptides as influenced by ripening temperatures and probiotic adjuncts in Cheddar cheeses", 《FOOD SCIENCE AND TECHNOLOGY》 * |
| YAN JIN等: "Peptide profiling and the bioactivity character of yogurt in the simulated gastrointestinal digestion", 《JOURNAL OF PROTEOMICS》 * |
| 于洋等: "乳源生物活性肽研究进展", 《食品与发酵工业》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112694429A (en) * | 2020-12-29 | 2021-04-23 | 江苏医药职业学院 | Polypeptide and application thereof in preparing ACE inhibitor or blood pressure lowering product |
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