CN106554387A - Nonapeptide with ACE and DPP-IV dual restraining activities and its application - Google Patents
Nonapeptide with ACE and DPP-IV dual restraining activities and its application Download PDFInfo
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- CN106554387A CN106554387A CN201510621748.8A CN201510621748A CN106554387A CN 106554387 A CN106554387 A CN 106554387A CN 201510621748 A CN201510621748 A CN 201510621748A CN 106554387 A CN106554387 A CN 106554387A
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Abstract
The present invention relates to one kind comes from the caseic polypeptide with Angiotensin-Converting (angiotensin-converting enzyme, ACE) and DPP-IV dual restraining activities.Its aminoacid sequence is Pro-Pro-Phe-Leu-Gln-Pro-Glu-Val-Met.Polypeptide PPFLQPEVM has ACE inhibitory activity and hypotensive activity, also there is DPP-IV inhibitory activity and hypoglycemic activity simultaneously, have a good application prospect as the health product and lead compound of the disease such as hypertension, heart disease and cardiovascular diseasess, diabetes, obesity, nephropathy, immunologic derangement.
Description
Technical field
The present invention relates to polypeptide PPFLQPEVM is preparing Angiotensin-Converting (ACE)
Inhibitor, dipeptidyl peptidase-IV (DPP-IV) inhibitor and blood pressure lowering, blood sugar lowering
Thing and health product.
Background technology
1.ACE inhibitory activity
Hypertension is a kind of common cardiovascular disease, and sickness rate height is to cause the 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.
2.DPP-IV inhibitory activity
Dipeptidyl peptidase-IV (dipeptidyl peptidase-IV, DPP-IV) is (EC3.4.14.5)
It is a kind of serine protease, is distributed widely in human body.DPP-IV is by cutting to polypeptide
Cutting inactivates which, so as to reach the effect of regulation of physiological functions.DPP-IV selective hydrolysises N- is last
There is on the second of end the protein of proline (Pro) or alanine (Ala).Its substrate specificity
Including：Glucagon-like-peptide-1 (GLP217-36), Gastric inhibitory polypeptide (GIP1-42), nerve
Peptide (NPY), YY peptides (PYY) and pancreatic polypeptide family (PP-family) etc..These materials
Common feature is exactly the deputy amino acid residue proline in N- ends or the third ammonia
Sour residue.DPP-IV by act on above peptide substrate and diabetes, glucose tolerance,
Obesity, appetite stimulator, hyperlipidemia, osteoporosises, neuropeptide metabolism and T- cells swash
Play an important role in the relevant disease such as living.Therefore, giving DPP-IV inhibitor in vivo can
The N- terminal degradations of prevention related substrates peptide, so as to ensure that function of human body is normally run.
Complete GLP-1 and GIP reactions are cut off N- ends 2 amino acid residue by DPP-IV
Afterwards so as to which inactivation is so as to physiological reactions such as the blood glucose regulations that affects GLP-1 related to GIP.
The N- terminal degradations that DPP-IV inhibitor can prevent GLP-1 and GIP are given in vivo, make pancreas
Island element secretions increase so as to improve glucose tolerance.In view of DPP-IV inhibitor and blood glucose generation
The substantial connection thanked, DPP-IV inhibitor have become the research heat of new type 2 diabetes mellitus medicine
Point.
The content of the invention
It is an object of the invention to provide polypeptide PPFLQPEVM is suppressing ACE activity and drop blood
Pressure and suppression DPP-IV activity and the application in blood sugar lowering；Polypeptide PPFLQPEVM has
ACE inhibitory activity, DPP-IV inhibitory activity and blood pressure lowering, hypoglycemic activity, as height
The disease such as blood pressure, heart disease and cardiovascular diseasess, diabetes, obesity, nephropathy, immunologic derangement
Health product and lead compound have a good application prospect.
For achieving the above object, the present invention suppresses by ACE of the polypeptide PPFLQPEVM
The active ingredient of agent, DPP-IV inhibitor and blood pressure lowering, hypoglycemic activity.
Which has sequence table SEQ ID NO：Aminoacid sequence in 1；Polypeptide PPFLQPEVM
For ACE inhibitor, DPP-IV inhibitor and blood pressure lowering, hypoglycemic drug and health product
Active component, wherein pharmaceutically acceptable carrier or adjuvant can be added.
With ACE inhibitory activity, DPP-IV inhibitory activity and blood pressure lowering, hypoglycemic activity
Polypeptide PPFLQPEVM, aminoacid sequence is
Pro-Pro-Phe-Leu-Gln-Pro-Glu-Val-Met, is single-stranded linear structure, white powder,
Soluble in water, molecular weight is 1057.5Da；To ACE activity with acting on compared with high inhibition, IC50
For 34.63 μM；There is very strong inhibitory action to DPP-IV activity, IC50 is 0.44mM.
Polypeptide PPFLQPEVM 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 dipeptides of polypeptide PPFLQPEVM is
Aminoacid, respectively Val (hydrophobic) and Met (hydrophobic) are stated, N-terminal is hydrophobic amino acid
Pro, therefore fully meet requirement.
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.The hydrophobic ammonia of polypeptide PPFLQPEVM
Base acid has Pro, Pro, Phe, Leu, Pro, Val and Met respectively, containing more hydrophobic
Aminoacid.
The present invention compared with prior art, has the advantages that：
The present invention is obtained from casein first and determines the structure of reactive compound, compound
With the activity for preferably suppressing ACE and DPP-IV, therefore as hearts such as treatment hypertension
The guarantor of the disease such as angiopathy and diabetes, heart disease, obesity, nephropathy, immunologic derangement
Strong product and lead compound have good potentiality and application prospect.
Specific embodiment
The identification of 1 polypeptide PPFLQPEVM 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 and DPP-IV.
Its concrete grammar is as follows：Sample and 35mM NaCl solutions are according to 1：1 volume ratio is mixed
Close, be 2.0 with 1M salt acid for adjusting pH, add pepsin, addition is pepsin：
Albumen quality ratio=1 in sample:100,1h is digested in 37 DEG C.1M NaOH adjust pH to 7.0
Terminate reaction.Enzymolysis solution enzymolysis solution turns in 4 DEG C, 3000～10000g (3000～10000g)
Speed lower 20 minutes 10-60 minutes of centrifugation) reactant liquor, supernatant molecular cut off is 10000
Ultrafilter membrane (3000～10000) ultrafiltration, filtrate is with C18-SPE post desalinations.Will after desalination
Enzymolysis sample lyophilizing, is dissolved in 0.1% formic acid water of mass concentration, LTQ Orbitrap XL (ions
Trap cyclotron resonance combines mass spectrograph) mass spectral analyses are carried out to sample.
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 PPFLQPEVM.
The ACE inhibitory activity detection of 2 polypeptide PPFLQPEVM of embodiment
Principle
The simulation bottom of ACE catalytic decomposition angiotensin Is under conditions of 37 DEG C, PH8.3
Thing Hippuryl-L-Histidyl-L-Leucine (HHL) produces hippuric acid, and the material is in 228nm
There is feature ultraviolet absorption peak at place.When ACE mortifiers are added, catalysis of the ACE to HHL
Effect is suppressed, and the growing amount of hippuric acid can be reduced.Before and after determining addition inhibitor
Hippuric acid ultraviolet absorption value 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 PPFLQPEVM obtained using embodiment 1 is operated as sample.
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, ACE is carried out to polypeptide PPFLQPEVM as stated above
Inhibitory activity is detected.As a result it is as follows：
The DPP-IV inhibitory activity detection of 3 polypeptide PPFLQPEVM of embodiment
Principle
Adopt the color development bottom with glycyl proline paranitroanilinum (Gly-Pro-PNA) as substrate
Thing method screens DPP-IV inhibitor, and the Cleaning Principle of the method is DPP-IV in the basic conditions
Catalytic substrate Gly-Pro-p-nitroanilide is hydrolyzed, and generates the paranitroanilinum of yellow, Hou Zhe
There is characteristic absorption peak at wavelength 405nm, the extinction measured at 405nm by microplate reader
Degree size reflection enzymatic activity height.
Method
Sample：Sample is dissolved in into the Tris-HCl buffer (pH=8.0) of 100mM, is matched somebody with somebody
Into 40mg/mL storing solutions, then storing solution is diluted to into different concentration, as sample solution.
Substrate：Gly-pro-p-nitroanilide solution, with the Tris-HCl buffer of 100mM
(pH=8.0) Gly-pro-p-nitroanilide is configured to into 1.59mM's
Gly-pro-p-nitroanilide solution.
Positive drug：Diprotin solution As, Diprotin A are delayed with the Tris-HCl of 100mM
Rush liquid (pH=8.0) and be configured to 2mM storing solutions, using being front diluted to 800uM.It is remaining
If storing solution is distributed into main, -20 DEG C of preservations.
Enzyme：DPP-IV solution, is matched somebody with somebody with the Tris-HCl buffer (pH=8.0) of 100mM
It is set to the DPP-IV solution of 0.01U/mL.
Stop bath：Acetic acid-the sodium-acetate buffer (pH=4.0) of 1M.
Experiment is carried out in 96 orifice plates, detects absorbance in 405nm using microplate reader.First
By enzyme, buffer and medicine at a temperature of 37 DEG C distinguish water-bath 30min, then by sample (or
Buffer), substrate sequentially add in 96 orifice plates 37 DEG C and be incubated 10 minutes, add DPP-IV
Enzymatic solution, mixes and incubates 60 minutes after 37 DEG C, adds the acetic acid-acetic acid of 100 μ L 1M
Sodium buffer (pH 4.0) makes reaction terminating.405nm absorbances are determined with microplate reader.Reaction
Cumulative volume is 100 μ l.Experiment is divided into 4 groups, and 3 multiple holes are set per group.Each group is respectively：
Sample sets (S groups)：Sample+enzyme+substrate.
Sample blank group (SB groups)：Sample+substrate.
Negative control group (C groups)：Enzyme+substrate.
Blank group (B groups)：Substrate.
Concrete each group sample-adding product are shown in Table 1.
1 DPP-VI inhibitory activity experiment packet of table and sample-adding amount
Note：(1) in table, the unit of numeral is μ l.
(2) cumulative volume is reacted for 100 μ l, each group is added after reactant with buffering according to listed in table
Liquid mends final volume to 100 μ l.
(3) calculating of suppression ratio
S group suppression ratio=(1-) × 100%
(4) testing result
Respectively with 30 μ g/ml, 60 μ g/ml, 120 μ g/ml, 300 μ g/ml, 600 μ g/ml,
The peptide concentration of 1200 μ g/ml, carries out DPP-IV inhibitory activity detections as stated above.Knot
Fruit such as following table：
Jing IC50Computer calculates the IC of the sequence50For 0.464mg/mL, i.e. 0.439mM.
Claims (4)
1. there is the nonapeptide of ACE and DPP-IV dual restraining activities, it is characterised in that：It is described
Nonapeptide is PPFLQPEVM, with sequence table SEQ ID NO：Aminoacid sequence in 1；
The aminoacid sequence of the polypeptide is specially Pro-Pro-Phe-Leu-Gln-Pro-Glu-Val-Met.
2. nonapeptide described in a kind of claim 1 prepare ACE inhibitor or Altace Ramipril, with
And in DPP-IV inhibitor or hypoglycemic drug is prepared during one or two or more kinds should
With.
3. according to the application described in claim 2, it is characterised in that：The ACE inhibitor
With in Altace Ramipril and DPP-IV inhibitor and hypoglycemic drug one or two or more kinds be
With nonapeptide PPFLQPEVM as active ingredient.
4. according to the application described in Claims 2 or 3, it is characterised in that：The ACE suppressions
In preparation and Altace Ramipril and DPP-IV inhibitor and hypoglycemic drug it is a kind of or two kinds with
Pharmaceutically acceptable carrier or adjuvant can be added in upper.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510621748.8A CN106554387B (en) | 2015-09-25 | 2015-09-25 | Nonapeptide with ACE and DPP-IV double inhibition activity and application thereof |
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| CN201510621748.8A CN106554387B (en) | 2015-09-25 | 2015-09-25 | Nonapeptide with ACE and DPP-IV double inhibition activity and application thereof |
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| CN106554387B CN106554387B (en) | 2020-01-21 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112921062A (en) * | 2021-03-16 | 2021-06-08 | 华南理工大学 | Casein peptide capable of reducing blood sugar in non-insulin-dependent way and preparation method and application thereof |
| CN114456232A (en) * | 2021-11-09 | 2022-05-10 | 中国科学院动物研究所 | Medicine for inhibiting diet obesity and polypeptide used by same |
| WO2024071404A1 (en) * | 2022-09-30 | 2024-04-04 | 森永乳業株式会社 | Novel peptide, and antioxidant composition |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1623600A (en) * | 2003-12-04 | 2005-06-08 | 中国科学院大连化学物理研究所 | Inhibitor of angiotensin I transferase activity and its application |
| WO2008108285A1 (en) * | 2007-03-02 | 2008-09-12 | Snow Brand Milk Products Co., Ltd. | Peptide |
| CN101618207A (en) * | 2009-06-17 | 2010-01-06 | 中国科学院大连化学物理研究所 | Application of polypeptide in preparing ACE inhibitor and medicine for lowering blood pressure |
-
2015
- 2015-09-25 CN CN201510621748.8A patent/CN106554387B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1623600A (en) * | 2003-12-04 | 2005-06-08 | 中国科学院大连化学物理研究所 | Inhibitor of angiotensin I transferase activity and its application |
| WO2008108285A1 (en) * | 2007-03-02 | 2008-09-12 | Snow Brand Milk Products Co., Ltd. | Peptide |
| CN101618207A (en) * | 2009-06-17 | 2010-01-06 | 中国科学院大连化学物理研究所 | Application of polypeptide in preparing ACE inhibitor and medicine for lowering blood pressure |
Non-Patent Citations (2)
| Title |
|---|
| EVANTHIA MONOGIOUDI等: "Effect of enzymatic cross-linking of b-casein on proteolysis by pepsin", 《FOOD HYDROCOLLOIDS》 * |
| YAN JIN等: "Peptide profiling and the bioactivity character of yogurt in the simulated gastrointestinal digestion", 《JOURNAL OF PROTEOMICS》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112921062A (en) * | 2021-03-16 | 2021-06-08 | 华南理工大学 | Casein peptide capable of reducing blood sugar in non-insulin-dependent way and preparation method and application thereof |
| CN114456232A (en) * | 2021-11-09 | 2022-05-10 | 中国科学院动物研究所 | Medicine for inhibiting diet obesity and polypeptide used by same |
| WO2023159866A1 (en) * | 2021-11-09 | 2023-08-31 | 中国科学院动物研究所 | Drug for inhibiting dietary obesity and polypeptide used thereby |
| CN114456232B (en) * | 2021-11-09 | 2023-09-29 | 中国科学院动物研究所 | Medicine for inhibiting edible obesity and polypeptide used by medicine |
| WO2024071404A1 (en) * | 2022-09-30 | 2024-04-04 | 森永乳業株式会社 | Novel peptide, and antioxidant composition |
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| Publication number | Publication date |
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| CN106554387B (en) | 2020-01-21 |
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