CN104558113B - The polypeptide compound for coming from rape pollen and application with ACE inhibitory activity - Google Patents
The polypeptide compound for coming from rape pollen and application with ACE inhibitory activity Download PDFInfo
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- CN104558113B CN104558113B CN201310507799.9A CN201310507799A CN104558113B CN 104558113 B CN104558113 B CN 104558113B CN 201310507799 A CN201310507799 A CN 201310507799A CN 104558113 B CN104558113 B CN 104558113B
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- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 15
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- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
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- 239000002327 cardiovascular agent Substances 0.000 claims 1
- 229940125692 cardiovascular agent Drugs 0.000 claims 1
- 102100030988 Angiotensin-converting enzyme Human genes 0.000 abstract description 35
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- AAXWBCKQYLBQKY-IRXDYDNUSA-N (2s)-2-[[(2s)-2-[(2-benzamidoacetyl)amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-4-methylpentanoic acid Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)CNC(=O)C=1C=CC=CC=1)C1=CN=CN1 AAXWBCKQYLBQKY-IRXDYDNUSA-N 0.000 description 4
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- CUKWUWBLQQDQAC-VEQWQPCFSA-N (3s)-3-amino-4-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2s,3s)-1-[[(2s)-1-[(2s)-2-[[(1s)-1-carboxyethyl]carbamoyl]pyrrolidin-1-yl]-3-(1h-imidazol-5-yl)-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-methyl-1-ox Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C1=CC=C(O)C=C1 CUKWUWBLQQDQAC-VEQWQPCFSA-N 0.000 description 2
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Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to come from having for rape pollen to suppress Angiotensin-Converting（angiotensin‑converting enzyme,ACE）The polypeptide compound IIIALLLYK of activity, its amino acid sequence is respectively IIe IIe IIe Ala Leu Leu Leu Tyr Lys.Polypeptide IIIALLLYK has ACE inhibitory activity, and the health products and lead compound as hypertension, heart disease and cardiovascular disease have a good application prospect.
Description
Technical field
The present invention relates to the polypeptide compound with ACE inhibitory activity of polypeptide rape pollen, it is characterised in that：It is described more
Peptide is that IIIALLLYK prepares Angiotensin-Converting（ACE）The application of inhibitor.
Background technology
One target spot of hypertension drug research is to suppress angiotensin converting enzyme（angiotensin-converting
enzyme,ACE）, ACE is present in vascular endothelial cell, epithelium absorption cell, neuro-epithelial cell, male sex-cell
A kind of dipeptides carboxylic acid containing zinc.In human body renin-angiotensin system（RAS）With kassinin kinin enzyme r e lease enzyme-kinin system
（KKS）In, Angiotensin-Converting（ACE）Important adjustment effect is played to body blood pressure and cardiovascular function（Such as Fig. 1）.
In RAS, ACE has been speed limit effect, and ACE can be angiotensin I converting for strong contraction blood by not active decapeptide
The octapeptide Angiotensin II of pipe effect, while Angiotensin II can also stimulate the secretion of aldosterone and then promote kidney pair
The reabsorption of water, sodium, potassium, cause the increase of sodium reserves and blood volume, raise blood pressure.In KKS, ACE can also be catalyzed and the heart
Vascular system has the degraded of neuropeptide, enkephalins and the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 of interaction etc..Angiotensin-Converting（ACE）Suppress
Agent is one of six major class antihypertensive drugs routinely applied at present, and Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe is used not only for treating hypertension, had proven to simultaneously
Heart failure can be prevented, reduces the danger that myocardial infarction, cerebral apoplexy, diabetes and renal damage occur.Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe is to painstaking effort
The preventing and treating of pipe disease has two obvious features：Definite efficacy of antihypertensive treatment and unique heart and vascular protection.Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe
It may be reconstructed by preventing the electricity in atrium from reconstructing and dissect, prevent atrial fibrillation（Atrial fibrillation, AF）Generation.Polypeptide is a kind of weight
The Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe wanted, the Peptides of native protein are the main sources of Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe peptide（Document 2：Lieselot
Vercruysse,John Van Camp,Guy Smagghe,J.Agric.Food Chem2005,53:8106-8115）.Blood vessel
Angiotensin-converting enzyme plays important adjustment effect for body blood pressure and cardiovascular function, therefore suppresses the medicine of ACE activity
Played a significant role in the treatment of the diseases such as angiocarpy, heart failure.The Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe of polypeptide while hypotensive not
The side effects such as the dry cough of common antihypertensive drugs can be caused.
The content of the invention
It is an object of the invention to provide applications of the polypeptide IIIALLLYK in ACE activity is suppressed；Polypeptide IIIALLLYK has
There is ACE inhibitory activity, the health products and lead compound as hypertension, heart disease and cardiovascular disease have good answer
Use prospect.
To achieve the above object, the present invention is using the polypeptide IIIALLLYK as the effective ingredient for suppressing ACE activity.It has
Ordered list SEQ ID NO：Amino acid sequence in 1；Polypeptide IIIALLLYK is the active ingredient of Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, wherein can add
Pharmaceutically acceptable carrier or auxiliary material.
Amino acid sequence with the polypeptide compound IIIALLLYK for suppressing ACE activity is IIe-IIe-IIe-Ala-
Leu-Leu-Leu-Tyr-Lys.Single-stranded linear structure, molecular weight 1059.4Da, white powder is soluble in water, and ACE is lived
Property has very strong inhibitory action, IC50For 28.2 μM.
Polypeptide IIIALLLYK possesses the feature required by Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe：
1.ACE tends to the substrate or inhibitor that hydrophobic amino acid is contained in each position of C-terminal tripeptides, and C-terminal is
Try, Phe, Tyr and Pro and N-terminal have stronger ACE inhibitory activity for the peptide fragment of branched-chain amino acid.Polypeptide IIIALLLYK C
End tripeptides is that hydrophobic amino acid is respectively Leu, Tyr and Lys, and N-terminal IIe is branched amino acid, therefore
IIIALLLYK meets the required peptide structural requirement of Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe.
2. the hydrophobicity of peptide is to influence the major reason of its inhibitory activity, the high peptide of inhibitory activity is all containing more hydrophobic
Amino acid.Polypeptide IIIALLLYK hydrophobic amino acid has Ala, Val, Leu, Tyr, Lys, IIe, contains more hydrophobic amino
Acid.
Detected through ACE inhibitory activity, polypeptide IIIALLLYK IC50For 28.2 μM.
The present invention compared with prior art, has the advantages that：
The present invention obtains from rape pollen and the structure of reactive compound is determined first, and compound has preferably suppression
ACE processed activity, therefore have as the lead compound of the cardiovascular disease medicines such as treatment hypertension, heart disease and health products
Good potentiality and application prospect.
Brief description of the drawings
Adjustment effect block diagrams of Figure 1A CE to blood pressure.
Embodiment
The polypeptide IIIALLLYK of embodiment 1 preparation
After 20g bee pollen form coles add liquid nitrogen grinding broken wall, pH=7.4,50mM Tris-HCl buffer solution 150ml are added,
Ultrasonic extraction is three times（Once：400W, ultrasonic 4s, stop 4s, 80 circulations）；Extraction terminates, after 4 DEG C, 25000g centrifugation 1h, to take
Supernatant, add the mixed solvent of 4 times of volumes of supernatant（Acetone：Ethanol：Acetic acid=50:50:0.1（Volume ratio））Precipitates overnight；
Precipitation centrifuges all samples 30 minutes in 4 DEG C, 25000g after terminating, and supernatant discarding, takes precipitation to wash decolouring with acetone, then
In 4 DEG C, 25000g centrifuge 15 minutes, supernatant discarding, precipitation continue to be washed repeatedly with acetone until supernatant it is colourless, finally use volume
The ethanol of concentration 75% washing precipitation simultaneously centrifuges 10 minutes in 4 DEG C, 25000g, abandons supernatant, taking precipitate freeze-drying, obtains lyophilized egg
In vain.PH=7.4 of the lyophilized protein containing 8M urea, 100mM NH4HCO3 buffer solutions redissolve, with enzyme：Albumen=1:25（w/w）Than
Example adds trypsase, and 37 DEG C digest 20 hours.Above-mentioned enzymolysis product is through preparing chromatographic isolation, chromatographic column C18
（20X250mm, 10 μm）, mobile phase A used is 0.1% formic acid of mass concentration/aqueous solution, and Mobile phase B is mass concentration 0.1%
Formic acid/acetonitrile solution, flow velocity 2mL/min, elution process is as follows：5%B—35%B(V/V)—80%B(V/V).Will be
The chromatographic peak detected under 280nm collects obtained different component freeze-drying respectively, is cream powder sample.
The inverted liquid chromatography-tandem mass spectrometry system of gained sample（RPLC-MS/MS）Analysis：By the enzyme after freeze-drying
Solution sample is formulated as 0.4 μ g/ μ L with the formic acid solution of volumetric concentration 0.1%, and the μ L of sample introduction 20 carry out LTQ linear ion hydrazine mass spectrums（Configuration
Nanoliter level electrospray ionization source）Analysis.System control and Data Collection are carried out using Xcalibur softwares (Thermo).What experiment obtained
Mass spectrometric data result is respectively in honeybee, Brassica campestris L. Protein Data Banks（http://
www.uniprot.org/）In retrieved, technology is repeated 3 times, database retrieval software SEQUEST.Obtaining sequence is
IIe-IIe-IIe-Ala-Leu-Leu-Leu-Tyr-Lys polypeptide.
The polypeptide IIIALLLYK of embodiment 2 ACE inhibitory activity detection
The simulation substrate Hippuryl-L- of angiotensin I is catalytically decomposed in ACE under conditions of 37 DEG C, PH8.3
Histidyl-L-Leucine (HHL) produces hippuric acid, and the material has feature ultraviolet absorption peak at 228nm.Press down when adding ACE
During thing processed, catalytic action of the ACE to HHL is suppressed, and the growing amount of hippuric acid can be reduced.Added by determining before and after inhibitor
Hippuric acid ultraviolet absorption value can calculate the size of inhibitory activity.
Reaction system
Buffer solution is 0.05M, PH8.3 borate buffer solutions；Substrate is Hippuryl-L-Histidyl-L-Leucine
(HHL), MW429.47,5mM is made into above-mentioned buffer solution；ACE（angiotensin-converting enzyme）Delayed with above-mentioned
Fliud flushing is made into 0.1U/ml.
ODA（Control group, for the light absorption value in the absence of inhibitor but when enzyme be present）：50 μ l buffer solution+50ulHHL+50 μ l
Then buffer solution adds 50 μ lACE, 37 DEG C of water-bath 30min in 37 DEG C of water-bath 5min, add 200 μ l, 1M HCl terminating reactions,
1ml ethyl acetate extraction production of hippuric acid is added, 15S is vibrated, 3500r/min centrifugation 5min, takes 0.8ml supernatants, 90 DEG C of water
15min is dried in bath, is dissolved in again in 0.8ml distilled water, and it is OD that light absorption value is detected at 228nmA。
ODB（Polypeptide sample group, light absorption value during inhibitor and enzyme to be present）:The μ lHHL+50 μ l of 50 μ l samples+50 are buffered
Then liquid adds 50 μ l ACE, 37 DEG C of water-bath 30min in 37 DEG C of water-bath 5min, add 200 μ l, 1M HCl terminating reactions, then
1ml ethyl acetate extraction production of hippuric acid is added, 15S is vibrated, 3500r/min centrifugation 5min, takes 0.8ml supernatants, 90 DEG C of water-baths
15min is dried, is dissolved in again in 0.8ml distilled water, it is OD that light absorption value is detected at 228nmB。
ODC（Blank group, light absorption value during in the absence of inhibitor and enzyme）：The μ lHHL+50 μ l of 50 μ l buffer solutions+50 are buffered
Then liquid adds 50 μ l buffer solutions, 37 DEG C of water-bath 30min, adds 200 μ l, 1M HCl terminating reactions in 37 DEG C of water-bath 5min,
1ml ethyl acetate extraction production of hippuric acid is added, 15S is vibrated, 3500r/min centrifugation 5min, takes 0.8ml supernatants, 90 DEG C of water
15min is dried in bath, is dissolved in again in 0.8ml distilled water, and it is OD that light absorption value is detected at 228nmC。
ACE inhibiting rates（%）=(ODA-ODB)/(ODA-ODC)×100%
Use 0.07275mg/ml, 0.04850mg/ml, 0.02425mg/ml respectively, 0.01217mg/ml peptide concentration,
ACE inhibitory activity detection is carried out as stated above.As a result such as following table：
Through IC50Calculator calculates the IC of the sequence50For 29.88 μ g/mL, i.e., 28.2 μM.
Claims (3)
1. the polypeptide compound for coming from rape pollen with ACE inhibitory activity, it is characterised in that：The polypeptide is
IIIALLLYK, there is sequence table SEQ ID NO：Amino acid sequence in 1；The amino acid sequence of the polypeptide is specifically, IIe-IIe-
IIe-Ala-Leu-Leu-Leu-Tyr-Lys。
A kind of 2. application of the polypeptide in Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe or cardiovascular drugs is prepared described in claim 1.
3. according to the application described in claim 2, it is characterised in that：The Vel-Tyr-Pro-Trp-Thr-Gln-Arg-Phe is using polypeptide IIIALLLYK as activity
Composition, wherein pharmaceutically acceptable carrier or auxiliary material can be added.
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Citations (1)
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| CN101353687A (en) * | 2008-07-31 | 2009-01-28 | 中国农业科学院蜜蜂研究所 | Melissa powder peptides having angiotensin transferase inhibitory activity and preparation thereof |
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| CN101353687A (en) * | 2008-07-31 | 2009-01-28 | 中国农业科学院蜜蜂研究所 | Melissa powder peptides having angiotensin transferase inhibitory activity and preparation thereof |
Non-Patent Citations (4)
| Title |
|---|
| Ace inhibitory activity in enzymatic hydrolysates of insect protein;Vercruysse等;《J.Agric.Food.Chem.》;20051231;第53卷(第13期);5207-5211 * |
| 大米ACE抑制肽的研究;刘焕;《中国优秀博硕士学位论文全文数据库（硕士） 医药卫生科技辑》;20060915(第9期);全文 * |
| 植物ACE抑制肽与峰花粉ACE抑制肽的研究现状;李天娇 等;《食品科学》;20091231;第30卷(第15期);261-263 * |
| 油菜蜂花粉ACE抑制物的制备及分离;李天骄;《中国优秀硕士学位论文全文数据库 工程科技I辑》;20111015(第10期);第2-5章 * |
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