CN102532265A - Gramicidin, isomer thereof and application thereof - Google Patents
Gramicidin, isomer thereof and application thereof Download PDFInfo
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- CN102532265A CN102532265A CN2011104491297A CN201110449129A CN102532265A CN 102532265 A CN102532265 A CN 102532265A CN 2011104491297 A CN2011104491297 A CN 2011104491297A CN 201110449129 A CN201110449129 A CN 201110449129A CN 102532265 A CN102532265 A CN 102532265A
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Abstract
The invention discloses gramicidin, isomer thereof and application thereof. The structure is Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-NH2 and Ac-Asp-Arg-Val-Tyr(D)-Ile-His(D)-Pro. Two modified construction bodies have biological activity which is similar to angiotensin 1-7 and capable of promoting human umbilical vein endothelial cells (HUVEC) to release nitrogen oxide (NO) for migration, forming a tubelet and restraining A549 cell growth. Simultaneously, the modified construction bodies have degradation capability of three degradation enzymes including ACE, NEP and AP, thereby ensuring that the modified construction bodies have longer half-life periods than the angiotensin 1-7. The modified construction bodies are simple to obtain and provide important application value for development of novel polypeptide drugs for treating cardiovascular and cerebrovascular diseases.
Description
Technical field
The present invention relates to a kind of small peptide, its isomer and application thereof.Particularly relating to this small peptide, its isomer is vasodilator, hypotensive, antithrombotic, the control heart disorder of activeconstituents and the medicine that anticancer function is arranged.
Background technology
Aspartic acid-l-arginine-Xie Ansuan-tyrosine-Isoleucine-Histidine-proline(Pro) (Asp-Arg-Val-Tyr-Ile-His-Pro); Be abbreviated as DRVYIHP; It is that endogenous seven peptides also are called as ang1-7, is distributed widely in the body in the various tissues and body fluid.This seven peptide is mainly by 2 hydrolysis of Angiotensin II menses angiotensin saccharase, or synthetic on a small quantity and discharge by other approach of II Angiotensin II reason.Concentration in blood is generally the nmole level.The pharmacokinetic of ang1-7 finds that the ang1-7 vein is degraded after injecting in the body soon, and the transformation period is merely 8~12 seconds.Ang1-7 removes from blood plasma through four kinds of mechanism in human body: the 1. hydrolytic action of Zinc metallopeptidase Zace1 (ACE) mediation; 2. the 3. 4. renal glomerular filtration of hydrolytic action of leucine endopeptidase (LAP) mediation of the hydrolytic action of neutral endopeptidase (NEP) mediation.Wherein the hydrolytic action of enzymolysis mediation is the metabolic main path of ang1-7.
Ang1-7 is an important component part in the renin-angiotensin regulator control system.Angiotensin-converting enzyme 2 has reduced the content of Angiotensin II in blood being hydrolyzed to Angiotensin II ang1-7.The specific receptors Mas acceptor of ang1-7 is because of combining can directly to reduce the combination activity of AT1 to Angiotensin II after the ang1-7 activation simultaneously, and the ang1-7 long-time stimulus can reduce the AT1 receptor expression.Therefore the approach of angiotensin-converting enzyme 2 hydrolysis Angiotensin IIs generation ang1-7 is the main endogenous balance suppressor factor of balance adjustment Angiotensin II physiological function in the interior renin-angiotensin system of body in vivo.The Angiotensin II of endogenous generation is important virulence factor in multiple diseases of cardiovascular and cerebrovascular systems; Therefore except using ACE enzyme inhibitors (suppressing the generation < Puli's class>of ANGII) and its receptor blocking agent (mainly blocking AT1 receptor acting < husky smooth type >) now on the market in a large number, ANG1-7 has also included the people's sight scope in as the endogenous balance suppressor factor of ANGII.
Ang1-7 has the various biological activity as a kind of multi-functional physiological regulating control factor that is mainly balance Angiotensin II function.Early stage report, ang1-7 can promote endotheliocyte to generate NO and vasodilatory effect.Finding subsequently that ang1-7 has on cell is independent of AT
1Acceptor and AT
2Angiotensin receptor beyond the acceptor: Mas acceptor; And bring into play the BA of the nervous plain II function of multiple adjusting antagonizing vessel through this receptor, comprise a series of useful BAs such as anti-angiogenic hyperplasia, antithrombotic, anti-cardiac muscle fertilizer, arrhythmia.Ang1-7 also comes to light and has unique inhibition and VEGF in the relevant heteroplastic transplantation knurl effect of PIGF expression in addition.
Though ang1-7 has the characteristics of the resisting cardiovascular disease of highly significant, because behind its entering human body, the effect of plurality of enzymes degraded rapidly down in vivo descends its concentration greatly, has greatly limited its bioactive performance.
Summary of the invention
The present invention is a kind of small peptide and isomer thereof, and its structural formula (hereinafter to be referred as invention small peptide 1) is:
Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-NH
2
Wherein Ac-represents nitrogen terminated acetylated ,-NH
2Represent the one of carbon tip amidation.
(hereinafter to be referred as invention small peptide 2) structural formula of its isomer is:
Ac-Asp-Arg-Val-Tyr(D)-Ile-His(D)-Pro
Wherein " D " representes that this amino-acid residue is the D configuration.
The acquisition of this small peptide and isomer thereof adopts known method of the prior art to carry out, and can use conventional chemical synthesis process, or biosynthetic means obtains.
This small peptide and isomer thereof respectively with ACE enzyme, NEP enzyme, the effect of LAP enzyme, the HPLC check and analysis are confirmed it and can both be resisted this three kinds of enzyme liberating.With this small peptide and isomer thereof respectively antihypertensive drug, antithrombotic reagent and the cancer therapy drug of active ingredient; Have basic identical even be superior to the characteristic of natural ang1-7 with natural ang1-7; And resist above-mentioned three kinds of degrading enzymes in vivo and degrade; Containing under the enzyme condition its transformation period prolongs and surpasses prototype more than 100 times, makes the performance function that it more can continuous and effective.
This small peptide and isomer thereof can prepare like the vasodilator Altace Ramipril, and its active ingredient is small peptide of the present invention or its isomer of treatment significant quantity; Anti-angiogenic hyperplasia medicine, its active ingredient are small peptide of the present invention or its isomer of treatment significant quantity; Antiarrhythmic drug, its active ingredient are small peptide of the present invention or its isomer of treatment significant quantity; Cancer therapy drug, its active ingredient are small peptide of the present invention or its isomer of treatment significant quantity.
When needing, in said medicine, can also contain one or more pharmaceutically acceptable carriers.Said carrier comprises the conventional thinner of pharmaceutical field, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier, lubricant etc., can also add flavouring agent, sweeting agent etc. in case of necessity.
Medicine of the present invention can be processed tablet, pulvis, granula, capsule, and various ways such as lyophilized powder injection liquid.The medicine of above-mentioned various formulations all can be according to the ordinary method preparation of pharmaceutical field.
The consumption of said medicine is generally μ g/kg body weight every days 100.
Description of drawings
The HPLC of anti-hydrolysis result collection of illustrative plates for small peptide contrast hypertensin 1-7 of the present invention shown in Figure 1;
A figure: hypertensin 1-7 standard substance;
B figure: invention small peptide 1 standard substance;
C figure: invention small peptide 2 standard substance;
D figure: hypertensin 1-7 is by ACE enzymic hydrolysis result;
E figure: invention small peptide 1 is by ACE enzymic hydrolysis result;
F figure: invention small peptide 2 is by ACE enzymic hydrolysis result;
G figure: hypertensin 1-7 is by LAP enzymic hydrolysis result;
H figure: invention small peptide 1 is by LAP enzymic hydrolysis result;
I figure: invention small peptide 2 is by LAP enzymic hydrolysis result;
J figure: hypertensin 1-7 is by NEP enzymic hydrolysis result;
K figure: small peptide 1 of the present invention is by NEP enzymic hydrolysis result;
L figure: invention small peptide 2 is by NEP enzymic hydrolysis result.
The assay that stimulates HUVEC to discharge NO for small peptide 1 of the present invention shown in Figure 2 contrasts.
Shown in Figure 3 is hypertensin 1-7, small peptide of the present invention 1, small peptide of the present invention 2 inhibition HUVEC cells formation tubule comparison diagrams;
A figure is tubule number statistical figure;
B figure: the HUVEC cell forms visible tubule under the light microscopic under the no medicine irritation situation;
C figure: hypertensin 1-7 suppresses the HUVEC cell and forms visible tubule under the light microscopic;
D figure: small peptide 1 of the present invention suppresses the HUVEC cell and forms visible tubule under the light microscopic;
E figure: small peptide 2 of the present invention suppresses the HUVEC cell and forms visible tubule under the light microscopic.
Comparing result for small peptide inhibition A549 of the present invention cell proliferation shown in Figure 4, wherein:
A figure is that hypertensin 1-7 suppresses A549 cell growth result;
B figure is that small peptide 1 of the present invention suppresses A549 cell growth result;
C figure is that small peptide 2 of the present invention suppresses A549 cell growth result.
Embodiment
Implement the anti-hydrolysis result of 1 small peptide of the present invention and isomer thereof contrast hypertensin 1-7
Dissolve hypertensin 1-7, invention small peptide 1, invention small peptide 2 to 5mmol/L with PBS.
Dissolving hypertensin 1-7, invention small peptide 1, invention small peptide 25 μ l+20 μ l PBS 37 ℃ of water-bath 30min+5 μ l acetonitrile with PBS crosses HPLC and makes standard diagram.
Invention small peptide 15 μ l+10 μ l ACE or NEP or LAP enzyme (0.05U)+37 ℃ of water-bath 30min+5 of 10PBS μ l acetonitrile are crossed HPLC.
Invention small peptide 25 μ l+10 μ l ACE or NEP or LAP enzyme (0.05U)+37 ℃ of water-bath 30min+5 of 10PBS μ l acetonitrile are crossed HPLC.
Chromatographic condition
A liquid: 0.1% phosphoric acid solution
B liquid: 100% acetonitrile solution
C
18Post
Flow velocity: 0.35ml/min
Go up appearance behind the 12%B solution equilibria pillar, among the B liquid concentration 15min gradually gradient rise to 24%, 24%B liquid concentration continues flushing 15min.
The result sees Fig. 1, and is as shown in the figure, and than hypertensin 1-7 standard substance, two kinds of invention small peptides can better be resisted the degraded of degraded hypertensin 1-7 relevant enzyme.
Behind the digestion HUVEC cell with every hole 2 * 10
5Individual cell is spread into 12 orifice plates and is added DMEM37 ℃ of 5%C0 of 10%FBS
2Overnight cultures.
Get that 10%FBS DMEM dilutes ang1-7 respectively, its structural formula of small peptide of the present invention is Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-NH
2, and Ac-Asp-Arg-Val-Tyr (D)-Ile-His (D)-Pro-NH
2, be 1 μ m/L.12 holes add fresh culture or 37 ℃ of cultivations of medicine 900 μ l 30min after being divided into no medicine group, ang1-7, small peptide of the present invention 1,2, four groups every group 2 secondary holes absorption nutrient solutions of invention small peptide.
Taking out the every hole of 12 orifice plates gets 50 μ l nutrient solutions and does content of nitric oxide and measure.
The result sees Fig. 2, and is as shown in the figure, add modifier after modifier can stimulate vascular endothelial cell (HUVEC) to discharge nitrogen protoxide, and nitrogen protoxide is a kind of vasodilator active substance of strong effect and the body oxidative damage is had provide protection.
Precooling 96 orifice plates and 200 μ l rifle heads, the matrgiel matrigel of 4 ℃ of thawing-20 ℃ preservations of spending the night.
The every hole of matrgiel matrigel 50 μ l of melting is added 96 orifice plates of precooling fast, and room temperature is placed 10min and is treated that the matrgiel matrigel solidifies.
Digestion HUVEC cell is regulated cell density 2 * 10
5ML gets 144 μ l cell suspensions and adds on the matrgiel matrigel that solidifies.
DMEM allocates 1 μ m/L ang1-7, small peptide of the present invention 1, small peptide of the present invention 2,16 holes and is divided into 2 four groups of no medicine groups, ang1-7, small peptide of the present invention 1, small peptide of the present invention, and every group of 3 secondary holes add 16 μ l soups in 96 orifice plates that added cell.
37 ℃ of 5%CO
2Cultivating 8h observes tubule formation effect and calculates the tubule number.
The result sees Fig. 3; As shown in the figure; Small peptide of the present invention can form than the tubule of the better inhibition vascular endothelial cell of prototype; Endothelial cell migration, to merge and form tubular structure each other be the important step in the vascularization process, if can suppress this link, just can suppress the formation and the hyperplasia of blood vessel.
The every hole 1 * 10 of digestion A549 cell
4The shop is gone into 24 orifice plates and is added 10%FBS164037 ℃ of 5%CO
2Cultivate overnight cultures.
Hot pressing PBS (PH7.2) and with its dilution small peptide of the present invention.Take out cell counting in the digestion first row hole behind 24 orifice plates, cell is inhaled and is gone nutrient solution 4 row to add PBS respectively in all the other holes, and small peptide 0.1 μ m/L of the present invention, 1 μ m/L, 10 μ m/L cells washed are also hatched 30min at 37 ℃.
Suction goes washing lotion to add 164037 ℃ of 5%CO of 1%FBS
2Cultivate 1d.
Repeated second section step in second day, cell was also accomplished experiment up to the 6th day by that analogy in the digestion counting secondary series hole, did not have porocyte to count described point and drew cell growth curve.
The result sees Fig. 4, and is as shown in the figure, and small peptide of the present invention 1 and small peptide 2 of the present invention have the effect of propagation of the inhibition A549 cell (lung cancer) of similar Ang1-7.
Claims (11)
1. small peptide, its structural formula is:
Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-NH
2
Wherein Ac-represents nitrogen terminated acetylated ,-NH
2Represent the one of carbon tip amidation.
2. small peptide, its structural formula is:
Ac-Asp-Arg-Val-Tyr(D)-Ile-His(D)-Pro
Wherein " D " representes that this amino-acid residue is the D configuration.
3. vasodilator Altace Ramipril, its active ingredient are the said small peptide of claim 1 or the said small peptides of claim 2 of treatment significant quantity.
4. anti-angiogenic hyperplasia medicine, its active ingredient are the said small peptide of claim 1 or the said small peptides of claim 2 of treatment significant quantity.
5. antiarrhythmic drug, its active ingredient are the said small peptide of claim 1 or the said small peptides of claim 2 of treatment significant quantity.
6. cancer therapy drug, its active ingredient are the said small peptide of claim 1 or the said small peptides of claim 2 of treatment significant quantity.
7. said small peptide of claim 1 or the said small peptide of claim 2 application in making antihypertensive drugs.
8. said small peptide of claim 1 or the said small peptide of claim 2 application in making antiarrhythmic drug.
9. said small peptide of claim 1 or the said small peptide of claim 2 application in making antithrombotic reagent.
10. said small peptide of claim 1 or the said small peptide of claim 2 application in making cancer therapy drug.
11. contain in claim 7,8,9, the 10 said medicinal applications one or more pharmaceutically the acceptable carrier comprise made tablet such as the conventional thinner of pharmaceutical field, vehicle, weighting agent, tackiness agent, wetting agent, disintegrating agent, absorption enhancer, tensio-active agent, absorption carrier, lubricant, pulvis, granula, capsule, and medicinal forms and healthcare products and foodstuff additive form such as lyophilized powder injection liquid.
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103965297A (en) * | 2014-05-27 | 2014-08-06 | 上海第一生化药业有限公司 | Polypeptide as well as preparation method and application thereof |
CN106916204A (en) * | 2017-03-29 | 2017-07-04 | 佛山科学技术学院 | A kind of acetyl amide heptapeptide and its purification process and application |
CN107022000A (en) * | 2017-03-29 | 2017-08-08 | 佛山科学技术学院 | A kind of acetyl amide pentadecapeptide and its purification process and application |
CN110540577A (en) * | 2019-09-12 | 2019-12-06 | 浙江省农业科学院 | Duck source polypeptide with blood pressure lowering effect and application thereof |
CN110714028A (en) * | 2019-11-04 | 2020-01-21 | 中国人民解放军第四军医大学 | Controllable up-regulation Ang- (1-7) targeting expression vector for preventing and treating hypoxic pulmonary hypertension |
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WO2008056207A1 (en) * | 2006-11-08 | 2008-05-15 | Chongxi Yu | Transdermal delivery systems of peptides and related compounds |
CN102164614A (en) * | 2008-09-12 | 2011-08-24 | 柏林夏洛蒂医科大学 | Use of an Ang-(1-7) receptor agonist in acute lung injury |
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Patent Citations (2)
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WO2008056207A1 (en) * | 2006-11-08 | 2008-05-15 | Chongxi Yu | Transdermal delivery systems of peptides and related compounds |
CN102164614A (en) * | 2008-09-12 | 2011-08-24 | 柏林夏洛蒂医科大学 | Use of an Ang-(1-7) receptor agonist in acute lung injury |
Non-Patent Citations (3)
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J. SAMANEN ET AL.: "Effects of D-Amino Acid Substitution on Antagonist Activities of Angiotensin II Analogues", 《J. MED. CHEM.》, vol. 31, 31 December 1988 (1988-12-31), pages 1 - 1 * |
KENJI MIYAZAKI、AKIRA TSUGITA: "C-Terminal sequencing method for peptides and proteins by the reaction with a vapor of perfluoric acid in acetic anhydride", 《PROTEOMICS》, vol. 4, 31 December 2004 (2004-12-31), pages 1 - 11 * |
REGINA S.H. CARVALHO ET AL.: "Polystyrene-type resin used for peptide synthesis: application for anion-exchange and affinity chromatography", 《JOURNAL OF CHROMATOGRAPHY B》, vol. 817, 31 December 2005 (2005-12-31), pages 235 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103965297A (en) * | 2014-05-27 | 2014-08-06 | 上海第一生化药业有限公司 | Polypeptide as well as preparation method and application thereof |
CN103965297B (en) * | 2014-05-27 | 2016-08-17 | 上海第一生化药业有限公司 | One peptide species, its preparation method and application |
CN106916204A (en) * | 2017-03-29 | 2017-07-04 | 佛山科学技术学院 | A kind of acetyl amide heptapeptide and its purification process and application |
CN107022000A (en) * | 2017-03-29 | 2017-08-08 | 佛山科学技术学院 | A kind of acetyl amide pentadecapeptide and its purification process and application |
CN106916204B (en) * | 2017-03-29 | 2020-07-14 | 佛山科学技术学院 | Acetamidoated heptapeptide, and purification method and application thereof |
CN107022000B (en) * | 2017-03-29 | 2020-07-14 | 佛山科学技术学院 | Acetyl amidated pentadecapeptide, and purification method and application thereof |
CN110540577A (en) * | 2019-09-12 | 2019-12-06 | 浙江省农业科学院 | Duck source polypeptide with blood pressure lowering effect and application thereof |
CN110714028A (en) * | 2019-11-04 | 2020-01-21 | 中国人民解放军第四军医大学 | Controllable up-regulation Ang- (1-7) targeting expression vector for preventing and treating hypoxic pulmonary hypertension |
CN110714028B (en) * | 2019-11-04 | 2021-08-31 | 中国人民解放军第四军医大学 | Controllable up-regulation Ang- (1-7) targeting expression vector for preventing and treating hypoxic pulmonary hypertension |
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