CN104945502A - ACE (angiotensin converting enzyme) inhibitory pentapeptide - Google Patents
ACE (angiotensin converting enzyme) inhibitory pentapeptide Download PDFInfo
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- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 26
- UUUHXMGGBIUAPW-UHFFFAOYSA-N 1-[1-[2-[[5-amino-2-[[1-[5-(diaminomethylideneamino)-2-[[1-[3-(1h-indol-3-yl)-2-[(5-oxopyrrolidine-2-carbonyl)amino]propanoyl]pyrrolidine-2-carbonyl]amino]pentanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-methylpentanoyl]pyrrolidine-2-carbon Chemical compound C1CCC(C(=O)N2C(CCC2)C(O)=O)N1C(=O)C(C(C)CC)NC(=O)C(CCC(N)=O)NC(=O)C1CCCN1C(=O)C(CCCN=C(N)N)NC(=O)C1CCCN1C(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C1CCC(=O)N1 UUUHXMGGBIUAPW-UHFFFAOYSA-N 0.000 title abstract 4
- 102000004270 Peptidyl-Dipeptidase A Human genes 0.000 title abstract 4
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 title abstract 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 102000008186 Collagen Human genes 0.000 claims description 17
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- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- QXZGBUJJYSLZLT-FDISYFBBSA-N bradykinin Chemical compound NC(=N)NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(=O)NCC(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CO)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CCC1 QXZGBUJJYSLZLT-FDISYFBBSA-N 0.000 description 2
- FAKRSMQSSFJEIM-RQJHMYQMSA-N captopril Chemical compound SC[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O FAKRSMQSSFJEIM-RQJHMYQMSA-N 0.000 description 2
- 229960000830 captopril Drugs 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
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- 239000011734 sodium Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- PQSUYGKTWSAVDQ-ZVIOFETBSA-N Aldosterone Chemical compound C([C@@]1([C@@H](C(=O)CO)CC[C@H]1[C@@H]1CC2)C=O)[C@H](O)[C@@H]1[C@]1(C)C2=CC(=O)CC1 PQSUYGKTWSAVDQ-ZVIOFETBSA-N 0.000 description 1
- PQSUYGKTWSAVDQ-UHFFFAOYSA-N Aldosterone Natural products C1CC2C3CCC(C(=O)CO)C3(C=O)CC(O)C2C2(C)C1=CC(=O)CC2 PQSUYGKTWSAVDQ-UHFFFAOYSA-N 0.000 description 1
- 102000005862 Angiotensin II Human genes 0.000 description 1
- 101800000733 Angiotensin-2 Proteins 0.000 description 1
- 108010064733 Angiotensins Proteins 0.000 description 1
- 102000015427 Angiotensins Human genes 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010008190 Cerebrovascular accident Diseases 0.000 description 1
- 101000984728 Chiropsoides quadrigatus Angiotensin-converting enzyme inhibitory peptide Proteins 0.000 description 1
- FEWJPZIEWOKRBE-JCYAYHJZSA-N Dextrotartaric acid Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O FEWJPZIEWOKRBE-JCYAYHJZSA-N 0.000 description 1
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- 108010007979 Glycocholic Acid Proteins 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- CZGUSIXMZVURDU-JZXHSEFVSA-N Ile(5)-angiotensin II 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](CC=1C=CC=CC=1)C([O-])=O)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=[NH2+])NC(=O)[C@@H]([NH3+])CC([O-])=O)C(C)C)C1=CC=C(O)C=C1 CZGUSIXMZVURDU-JZXHSEFVSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
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- RFDAIACWWDREDC-UHFFFAOYSA-N Na salt-Glycocholic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCC(O)=O)C)C1(C)C(O)C2 RFDAIACWWDREDC-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 229960002478 aldosterone Drugs 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- -1 amine salt Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229950006323 angiotensin ii Drugs 0.000 description 1
- 210000002565 arteriole Anatomy 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
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- 159000000007 calcium salts Chemical class 0.000 description 1
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- RFDAIACWWDREDC-FRVQLJSFSA-N glycocholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 RFDAIACWWDREDC-FRVQLJSFSA-N 0.000 description 1
- 229940099347 glycocholic acid Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
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- 239000000463 material Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 230000029865 regulation of blood pressure Effects 0.000 description 1
- 230000036454 renin-angiotensin system Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
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- 235000014347 soups Nutrition 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides an ACE (angiotensin converting enzyme) inhibitory pentapeptide. An amino acid sequence of the pentapeptide is Gly-Ser-Val-Gly-Tyr(GSVGY). The short peptide has obvious antihypertensive effects. The active pentapeptide comes from food and also has the prominent advantages of high safety, low price and industrialization. The active pentapeptide or derivatives thereof can be used for preparing medicines for treating/preventing hypertension or serve as functional food additives for hypertension patients to use for the purpose of long-term treatment and health care.
Description
Technical field
The present invention relates to a kind of Zinc metallopeptidase Zace1 (ACE) inhibiting peptide and analogue thereof of biogenetic derivation.
Background technology
Hypertension is one of modal cardiovascular disorder, and it can cause the infringement of brain, cardiovascular, kidney, and be the important factor causing cerebral apoplexy, heart failure and coronary heart disease etc., the health of the mankind in serious threat.Therefore, treatment and preventing hypertension are to the healthy level improving the mankind, and prolongs life has great significance.Zinc metallopeptidase Zace1, in human body renin-angiotensin system and kallikrein kinin system, plays an important role to blood pressure regulation.Angiotensin i-converting can be angiotensinⅡ by ACE, makes around arteriole, vascular smooth muscle contraction, stimulates Aldosterone Secretion simultaneously, promotes that human kidney is to Na
+, K
+heavily absorption, cause the increase of sodium reserves and Q volume of blood, make elevation of blood pressure; Bradykinin inactivation can also be made, cause elevation of the blood pressure.In sum, ACE mono-aspect produces the angiotensin II making blood pressure increase, and on the other hand, make the bradykinin inactivation with vasorelaxation action, this all causes the rising of blood pressure.So, if inhibit the activity of ACE, the effect of step-down just can be played.
Existing as treating the inhibitor that hypertensive synthetics captopril is exactly ACE, but it has a lot of side effect, and so the ace inhibitory peptide come from food protein has no side effect because of it, have other curative effect simultaneously and be widely used, market outlook are fabulous.
Summary of the invention
The invention provides the bioactive peptide of a kind of separation from water-soluble fish scale collagen enzymolysis solution, this small peptide has Zinc metallopeptidase Zace1 (ACE) inhibit activities.
ACE of the present invention suppresses pentapeptide, and aminoacid sequence is Gly-Ser-Val-Gly-Tyr (GSVGY).
The ACE of the invention described above suppresses pentapeptide should comprise the pentapeptide being defined as L-type amino acid or the amino acid whose different configuration of D type by amino acid fine jade luminosity difference.
Preferably, ACE of the present invention suppresses pentapeptide to be derived from fish scale collagen.
On the other hand, the present invention also aims to provide the ACE described in the invention described above to suppress the preparation method of pentapeptide, comprise raw materials pretreatment, water-soluble collagen protein extraction and enzymolysis, and the step that active pentapeptide is separated, wherein said active pentapeptide is separated and comprises ultrafiltration, gel-filtration and C
18the step that high performance liquid chromatography is separated, the ACE inhibitory activity of tracing detection separated portion in sepn process, collects the component with ACE activity and carries out next step separation.
As one of concrete embodiment, the ACE of the invention described above to suppress described in the preparation method of pentapeptide and active pentapeptide be separated and comprise the steps:
(1) after enzymolysis product ultrafiltration, get molecular weight and be less than 3000 daltonian components, adopt Sephadex G-25 to be separated, moving phase is the methanol solution of 30%, and column temperature is room temperature, and determined wavelength is 280nm;
The ACE inhibitory activity of tracing detection separated portion, temporally collects the component V with ACE inhibitory activity;
(2) component V of step (1) gained is separated further through ion exchange chromatography, the ACE inhibitory activity of tracing detection separated portion, and collect the component Vb with ACE inhibitory activity, lyophilize is for subsequent use;
(3) the component Vb of step (2) gained is through PR-HPLC purifying, the ACE inhibitory activity of tracing detection separated portion, collects the component Vba with ACE inhibitory activity, lyophilize.
The ACE of the invention described above suppresses in the preparation method of pentapeptide, and in described step (2), component V is separated further through ion exchange chromatography, and obtain 3 components, temporally collect, intermediate component has ACE inhibitory activity, is component Vb.
The ACE of the invention described above suppresses in the preparation method of pentapeptide, and in described step (3), middle PR-HPLC condition comprises:
Hypersil C
18post; Determined wavelength 215nm, room temperature, sampling volume 20 μ l;
Gradient elution: 0 ~ 60min: mobile phase A, 100 ~ 50% linearly reduce;
Mobile phase B, 0 ~ 50% linearly raises;
60 ~ 80min: mobile phase A, 0%; Mobile phase B, 100%;
Wherein, mobile phase A is: according to volume percent, containing the 0.1%TFA aqueous solution, uses front ultrasonic degas;
Mobile phase B is: according to volume percent, containing the acetonitrile of 0.1%TFA, uses front ultrasonic degas.
One of further embodiment, the clear water that the ACE of the invention described above suppresses the raw materials pretreatment described in preparation method of pentapeptide to comprise the steps: dry fish scale to be placed in 10 ~ 20 times of weight soaks 2 ~ 4 hours, after filter screen removes free water content, be 0.1 ~ 1.0% salt acid soak 1 ~ 6 hour by the mass concentration of 10 ~ 12 times of weight again, then be that 0.1 ~ 0.5% sodium hydroxide solution is neutralized to neutrality with mass concentration, water elution is except salinity, filter screen drains, as water-soluble collagen protein extraction raw material fish scale.
In embodiment, the ACE of the invention described above suppresses the water-soluble collagen protein extraction described in preparation method of pentapeptide and enzymolysis to be that pretreated raw material fish scale and deionized water are loaded distilling kettle according to mass ratio 1:2 ~ 10,60 ~ 120 DEG C are heated 2 ~ 8 hours, and collecting solvent portions is water-soluble collagen protein; Then with stomach en-enzymolysis 24 hours.
In conjunction with the above-mentioned description of preparation method ACE being suppressed to pentapeptide, object of the present invention be also to protect by above-mentioned any one about preparation method technical scheme obtained by ACE suppress pentapeptide, or containing the composition obtained by described preparation method.
Again on the one hand, the present invention also aims to provide one to prevent and/or treat hypertensive compound, described composition contains ACE of the present invention and suppresses pentapeptide.Described composition comprises medicine or food compositions.
Based on fully understanding of the present invention above, applicant believes, those skilled in the art should easy understand, peptide of the present invention and by other related derivative product that universal method obtains, all should belong to the present invention protect prolong and category.Above-mentioned universal method citing but be not limited to produce salt with acid-respons, or the salt formed with metal/positively charged ion.Described acid includes but not limited to hydrochloric acid, sulfuric acid, nitric acid, the mineral acids such as phosphoric acid; Formic acid, acetic acid, propionic acid, glycocholic acid, oxysuccinic acid, citric acid, tartrate, the organic acids such as succsinic acid.The described salt formed with metal/positively charged ion includes but not limited to sodium salt, sylvite, calcium salt, ammonium salt, or and monoethanolamine, triethylamine, the amine salt etc. that two ring second ammonia etc. are formed.
On the other hand, to described in the present invention and the understanding of medicine, applicant believes, in conjunction with state of the art, described " medicine " unambiguously can be interpreted as various pharmaceutical preparation, includes but not limited to powder, granule, tablet, capsule, suppository, suspension, emulsion, spray, injection liquid or the powder injection etc. that add obtained by various medical auxiliary materials.According to different dosage form feature, when using these medicines to treat, can pass through various corresponding administration, typical but be not limited to can oral administration administration, drug administration by injection and mucosa delivery.
Again on the one hand, to described in the present invention and the understanding of food compositions, i.e. the food of ordinary meaning or healthcare products, form can be refreshment drink, lactic drink, seasonings, soup class, cheese, ham, dessert etc.
ACE that the present invention is correlated with suppresses pentapeptide or its correlated product, and usage quantity has no particular limits, and concrete will according to hypertensive degree, the age of patient, body weight, physical appearance and give with the factor such as method, suitably determine.
This pentapeptide has obvious blood pressure lowering effect.And this active pentapeptide is by food sources, have simultaneously high security, cheapness, can the outstanding advantages of industrialization.This active pentapeptide or derivatives thereof can be used for preparing hypertension therapeutic/prophylactic agent, or as function food additive, keeps healthy for hyperpietic's long-term treatment.
Embodiment
In the mode of specific embodiment, the invention will be further elaborated below.Without specified otherwise, the present invention is with reference to the ACE inhibitory activity of the method tracing detection separated portion of 102516358B.
1. the preparation of water-soluble collagen protein
Clear water dry fish scale being placed in 16 times of weight soaks 4 hours, after filter screen removes free water content, use the 0.5% salt acid soak 5 hours of 10 times of weight again, and then be neutralized to neutrality with 0.5% sodium hydroxide solution, water elution is except salinity, filter screen drains, as water-soluble collagen protein extraction raw material fish scale.
Raw material fish scale is loaded distilling kettle, adds the deionized water of 8 times of weight, 100 DEG C are heated 6 ~ 8 hours, and collecting solvent portions is water-soluble collagen protein.
2. fish scale collagen ACE suppresses the preparation of pentapeptide
In step 1, the water-soluble collagen protein of preparation is through stomach en-enzymolysis after 24 hours, enzymolysis product is through ultrafiltration, get molecular weight and be less than 3000 daltonian components, adopt Sephadex G-25, moving phase is the methanol solution of 30%, and column temperature is room temperature, and determined wavelength is 280nm, the ACE inhibitory activity of tracing detection separated portion, collects target components successively according to the time.
The component V with ACE inhibitory activity obtained after Sephadex G-25 is separated and component VI (Fig. 1), the wherein first wash-out of component V.
3.ACE suppresses the separation of pentapeptide
The further separation and purification of ion exchange chromatography is passed through through the separating obtained component of Sephadex G-25 V in step 2, and the ACE inhibitory activity of separating obtained cut is measured, result such as Fig. 2 demonstrates, component V has eluted 3 peaks after ion exchange column, according to time sequence, be followed successively by component Va, Vb and Vc, wherein component Vb has ACE inhibitory activity, and the ACE inhibitory activity of the cut at peak-peak place is 84.0%.
4.ACE suppresses purifying and the order-checking of pentapeptide
Be separated to step 3 the component Vb PR-HPLC obtained to be further purified, as shown in Figure 3, collect the component Vba with ACE inhibitory activity, lyophilize obtains product T-1 to separating resulting.
PR-HPLC condition comprises:
Hypersil C
18post; Determined wavelength 215nm, room temperature, sampling volume 20 μ l;
Gradient elution: 0 ~ 60min: mobile phase A, 100 ~ 50% linearly reduce;
Mobile phase B, 0 ~ 50% linearly raises;
60 ~ 80min: mobile phase A, 0%; Mobile phase B, 100%;
Wherein, mobile phase A is: according to volume percent, containing the 0.1%TFA aqueous solution, uses front ultrasonic degas;
Mobile phase B is: according to volume percent, containing the acetonitrile of 0.1%TFA, uses front ultrasonic degas.
Product T-1 is through protein sequence analyzer analysis, and sequence is Gly-Ser-Val-Gly-Tyr (GSVGY).
And record the 503nhibiting concentration IC of product T-1 to ACE
50for 0.045mol/L.
Embodiment 2. intravenous injection into animals is tested
Select 20 spontaneously hypertensive big white mouse to be experimental model, after big white mouse raises one week, be divided into 2 groups, each group 10, every day is by big white mouse body weight 20mg/kg dosage (middle dosage group) intravenous injection product T-1.
Result shows, after 1 week, the blood pressure of control group big white mouse continues to raise, and the blood pressure for the treatment of group spontaneously hypertensive big white mouse obviously declines, the average systolic of hypertension big white mouse drops to 160mmHg from 190mmHg and illustrates that product T-1 has obvious blood pressure lowering effect through intravenously administrable.
The experimentation on animals of embodiment 3. gavage
20 spontaneously hypertensive big white mouse are selected to be experimental model, after big white mouse raises one week, be divided into 3 groups, each group 10, every day is by big white mouse body weight 20mg/kg dosage (middle dosage treatment group) gavage product T-1, another group positive drug Kapp towing force (Captopril) control group, every day, result as shown in Figure 4 by big white mouse body weight 20mg/kg dosage gavage.
After 4 weeks, the blood pressure of control group big white mouse continues to raise, and the blood pressure for the treatment of group spontaneously hypertensive big white mouse obviously declines, the average systolic of hypertension big white mouse drops to 160mmHg from 185mmHg and illustrates that the administration of product T-1 oral administration has obvious blood pressure lowering effect, and with positive drug blood pressure lowering effect and trend basically identical.
Accompanying drawing explanation
Accompanying drawing 4 width of the present invention, wherein:
Fig. 1 is the separating spectrum of Sephadex G-25 to enzymolysis product;
Fig. 2 is the ion exchange chromatography collection of illustrative plates of component V;
Fig. 3 is the PR-HPLC separating spectrum of component Vb;
Fig. 4 is the blood pressure lowering effect of collagen peptide to hypertension big white mouse.
Claims (10)
1.ACE suppresses pentapeptide, and aminoacid sequence is Gly-Ser-Val-Gly-Tyr.
2. ACE according to claim 1 suppresses pentapeptide, it is characterized in that, comprises the pentapeptide being defined as L-type amino acid or the amino acid whose different configuration of D type by amino acid fine jade luminosity difference.
3. ACE according to claim 1 suppresses pentapeptide, it is characterized in that, is derived from fish scale collagen.
4. ACE according to claim 1 suppresses the preparation method of pentapeptide, comprises raw materials pretreatment, water-soluble collagen protein extraction and enzymolysis, and the step that active pentapeptide is separated, and it is characterized in that, described active pentapeptide is separated and comprises ultrafiltration, gel-filtration and C
18the step that high performance liquid chromatography is separated, the ACE inhibitory activity of tracing detection separated portion in sepn process, collects the component with ACE activity and carries out next step separation.
5. method according to claim 4, is characterized in that, described active pentapeptide is separated and comprises the steps:
(1) after enzymolysis product ultrafiltration, get molecular weight and be less than 3000 daltonian components, adopt Sephadex G-25 to be separated, moving phase is the methanol solution of 30%, and column temperature is room temperature, and determined wavelength is 280nm;
The ACE inhibitory activity of tracing detection separated portion, temporally collects the component V with ACE inhibitory activity;
(2) component V of step (1) gained is separated further through ion exchange chromatography, the ACE inhibitory activity of tracing detection separated portion, and collect the component Vb with ACE inhibitory activity, lyophilize is for subsequent use;
(3) the component Vb of step (2) gained is through PR-HPLC purifying, the ACE inhibitory activity of tracing detection separated portion, collects the component Vba with ACE inhibitory activity, lyophilize.
6. method according to claim 5, is characterized in that, in described step (2), component V is separated further through ion exchange chromatography, and obtain 3 components, temporally collect, intermediate component has ACE inhibitory activity, is component Vb.
7. method according to claim 5, is characterized in that, in described step (3), PR-HPLC condition comprises:
Hypersil C
18post; Determined wavelength 215nm, room temperature, sampling volume 20 μ l;
Gradient elution: 0 ~ 60min: mobile phase A, 100 ~ 50% linearly reduce;
Mobile phase B, 0 ~ 50% linearly raises;
60 ~ 80min: mobile phase A, 0%; Mobile phase B, 100%;
Wherein, mobile phase A is: according to volume percent, containing the 0.1%TFA aqueous solution, uses front ultrasonic degas;
Mobile phase B is: according to volume percent, containing the acetonitrile of 0.1%TFA, uses front ultrasonic degas.
8. method according to claim 4, it is characterized in that, described raw materials pretreatment comprises the steps: that clear water dry fish scale being placed in 10 ~ 20 times of weight soaks 2 ~ 4 hours, after filter screen removes free water content, be 0.1 ~ 1.0% salt acid soak 1 ~ 6 hour by the mass concentration of 10 ~ 12 times of weight again, be then that 0.1 ~ 0.5% sodium hydroxide solution is neutralized to neutrality with mass concentration, water elution is except salinity, filter screen drains, as water-soluble collagen protein extraction raw material fish scale.
9. method according to claim 4, it is characterized in that, described water-soluble collagen protein extraction and enzymolysis are that pretreated raw material fish scale and deionized water are loaded distilling kettle according to mass ratio 1:2 ~ 10, and 60 ~ 120 DEG C are heated 2 ~ 8 hours, and collecting solvent portions is water-soluble collagen protein; Then with stomach en-enzymolysis 24 hours.
10. prevent and/or treat hypertensive compound, suppress pentapeptide containing ACE according to claim 1.
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