CN103204909A - Antihypertensive active peptide VPPIPP (valine-proline-proline-isoleucine-proline-proline) - Google Patents

Abstract

The invention discloses an antihypertensive active peptide, wherein the amino acid sequence of the antihypertensive active peptide is valine-proline-proline-isoleucine-proline-proline. According to the antihypertensive active peptide disclosed by the invention, the antihypertensive capacity of synthesised hexapeptide is verified by selecting a preferable antihypertensive peptide structure on the basis of previous research, and via in-vitro biological activity and in-vivo biological activity. The synthesised hexapeptide with an antihypertensive capacity disclosed by the invention can be used for inhibiting the activity of ACE (angiotensin converting enzyme) and lowering the generation level of AngII respectively, so as to achieve the effect of reducing blood pressure. The antihypertensive active peptide has the characteristics of being high in efficiency and low in toxicity, has a good application prospect for developing medicines and functional foods for treating hypertension, and lays the foundation for future low-cost and high-yield preparation in medicine development because of being capable of prepared in a large scale via an artificial chemical synthesis technology.

Description

A kind of antihypertensive active peptide VPPIPP

Technical field

The present invention relates to a kind of antihypertensive active peptide, be specifically related to by with antihypertensive functional peptide section ILE-PRO-PRO (IPP) and the trans link of VAL-PRO-PRO (VPP) and a kind of antihypertensive synthetic six peptides by biological activity test checking in external and the body.

Background technology

The situation of China's hypertension morbidity is more and more severeer, the hypertensive medicine of current common treatment has following six kinds: diuretic(s) (may cause hypokalemia, influence carbohydrate metabolism, sugar tolerance is descended), beta-Blocking agent (can bring central nervous system, the untoward reaction of Digestive tract and vascular system), α-Zu Zhiji, calcium antagonist (can cause oedema, headache, flush, diuresis, ypotension and cardiac conduction are obstructed), angiotensin converting enzyme inhibitor (ACEI) and angiotensin-ii receptor retarding agent (can cause dry cough, blood potassium height, even angioedema).Because existing most of medicine has stronger side effect, so scientists is devoted to develop medicine or the food of high-efficiency low-toxicity always, such as saying, contain and suppress Zinc metallopeptidase Zace1 (angiotensin converting enzyme, ACE, EC3.4.15.1) functional food of Huo Xing little peptide or medicine are benefited the hyperpietic, and the present invention just is based on that this background is born.The ACE enzyme is a kind of exopeptidase, is the important factor of regulating blood pressure, mainly is present in the endotheliocyte of blood vessel, and content is the abundantest in the lung hair.According to document: Zhao Haizhen, Lu Zhaoxin, Liu Zhanmin etc. the progress [J] of the Zinc metallopeptidase Zace1 inhibiting peptide in whole food source. Chinese biochemical drug magazine, 2004,25 (5): 315-317 and document: Chen Qiang. angiotensin converting enzyme inhibitor and angiotensin II receptor antagonists combined utilization treatment renal glomerular disease [J]. nephropathy and dialysis renal transplantation magazine, 2001,10 (3): the report of 274-277, the main effect of ACE enzyme has two, one is to make angiotensin I (Ang I) be converted into Angiotensin II (Ang II), another one is to make the bradykinin inactivation, finishes the adjusting of blood pressure with these two function influence renin-angiotensin system (RAS).Angiotensin II can combine with the specific receptors AT1 in the vascular tissue, by the haemodynamics effect, make vasoconstriction, shrink glomerular arteriole,efferent, renal blood flow is descended, voltage rise height in the glomerular capillary, bradykinin can make vasorelaxation by the haemodynamics effect, the diastole glomerular arteriole,efferent, renal blood flow is risen, and the glomerular capillary internal drop is low.

Summary of the invention

First purpose of the present invention provides a kind of bioactive peptide that inhibition ACE enzymic activity realizes antihypertensive function that passes through of high-efficiency low-toxicity.

For achieving the above object, the technical solution used in the present invention is: a kind of antihypertensive active peptide, its aminoacid sequence is respectively: Xie Ansuan (Val)-proline(Pro) (Pro)-proline(Pro) (Pro)-Isoleucine (Ile)-proline(Pro) (Pro)-proline(Pro) (Pro) (being called for short VPPIPP).Bioactive peptide VPPIPP of the present invention has selected antihypertensive functional peptide section: IPP and VPP, and by approximate " palindrome " between trans link formation N-end and the C-end.Since Pro, the Phe that ACE enzyme C holds catalytic site to have is vulnerable to Ang I (DRVYIHPFHL) and the position of His group influence, and the C of VPPIPP holds the 4th amino acid also to be Pro, similar with Ang I.Therefore VPPIPP can have better hypertension ability than the IPPVPP of IPP and VPP cis link formation.

Second purpose of the present invention provides bioactive peptide VPPIPP in preparation prevention or treats application in hypertensive medicine or the food.

Thus, the 3rd purpose of the present invention provides a kind of antihypertensive composition, wherein contains bioactive peptide VPPIPP as effective constituent, and contains conventional pharmaceutical carrier and/or vehicle.

Active six peptide VPPIPP of the present invention can adopt this area ordinary method preparation, and are synthetic as adopting solid-phase peptide synthesis, specifically comprise following concrete steps:

At first insert the fluorenylmethyloxycarbonyl proline(Pro) at proline(Pro) dichloro resin; add TBTU and DIEA simultaneously and carry out coupling; coupling time is 30 minutes; and be that 20% piperidines/dimethyl formamide washing is removed terminal fluorenylmethyloxycarbonyl group 3 times with volume ratio; carrying out deprotection with hexahydropyridine then, is that piperidines/dimethyl formamide of 20% washs 6 times with volume ratio again after deprotection finishes.Drain the back and add the fluorenylmethyloxycarbonyl Isoleucine; TBTU and DIEA carry out coupling, and coupling time is 30 minutes, and wash 3 times with piperidines/dimethyl formamide of 20%; carrying out deprotection with hexahydropyridine, is that 20% piperidines/dimethyl formamide washing is drained for 6 times with volume ratio again.Drain the back and add the fluorenylmethyloxycarbonyl proline(Pro); TBTU and DIEA carry out coupling, and coupling time is 30 minutes, and wash 3 times with piperidines/dimethyl formamide of 20%; carrying out deprotection with hexahydropyridine, is that 20% piperidines/dimethyl formamide washing is drained for 6 times with volume ratio again.Drain the back and add the fluorenylmethyloxycarbonyl proline(Pro); TBTU and DIEA carry out coupling, and coupling time is 30 minutes, and wash 3 times with piperidines/dimethyl formamide of 20%; carrying out deprotection with hexahydropyridine, is that 20% piperidines/dimethyl formamide washing is drained for 6 times with volume ratio again.Drain the back and add the fluorenylmethyloxycarbonyl Xie Ansuan, TBTU and DIEA carry out coupling, coupling time is 30 minutes, and with 20% piperidines/dimethyl formamide washing 3 times, carry out deprotection with hexahydropyridine, be that 20% piperidines/dimethyl formamide washing is drained for 6 times with volume ratio again, at last by half preparation type RPLC (reversed-phase column: VYDAC-C18 post (4.6 * 250mm, 5 μ m); Linear gradient: solution A (being dissolved in the 0.1%(v/v of analytical pure level acetonitrile) trifluoroacetic acid), gradient: 5%-30%(v/v) 25 minutes, 100%(v/v) 25.1 minutes, stop 30 minutes; Solution B (being dissolved in the 0.1%(v/v of pure water) trifluoroacetic acid), gradient: 95%-70%(v/v) 25 minutes, 0%25.1 minute, stop 30 minutes; Flow velocity: 1.0 ml/min; The separated and collected elution peak, standby after the freeze-drying.

Six peptide VPPIPP of the present invention are based on that two kinds of tripeptides IPP being used widely and VPP are trans to be linked into, and have hypertension ability preferably, are verified by biological activity in external and the body.Synthetic six peptides with hypertension ability that the present invention obtains can be used in the activity that suppresses angiotensin-converting enzyme (ACE enzyme), reduce the generation level of Angiotensin II (Ang II), reach the effect that brings high blood pressure down.This antihypertensive active peptide has characteristics of high efficiency and low toxicity, for exploitation treatment hypertension drug and functional food application promise in clinical practice is arranged.Owing to can carry out mass preparation by artificial chemical synthesising technology, for preparation low-cost and high yield in the future drug development is laid a good foundation.

Description of drawings

Fig. 1 for rat feed Isoleucine (Ile)-proline(Pro) (Pro)-proline(Pro) (Pro) (being called for short IPP) (1.5mg/kg) post shrinkage press that (Systolic Blood Pressure is SBP) with time relation figure;

Fig. 2 feeds VPPIPP(1.5mg/kg for rat) (Systolic Blood Pressure is SBP) with time relation figure for the post shrinkage pressure;

Fig. 3 is that (Systolic Blood Pressure is SBP) with time relation figure for blank group rat systolic pressure.

Embodiment

Below further set forth the present invention.

The test of six peptide molecule VPPIPP antihypertensive actives:

Experiment material:

New zealand white rabbit: pharmaceutical college of Shanghai Communications University experimentation on animals center provides; 20 all male hypertension pattern rats in age (SHR): the Shanghai Experimental Animal Center provides; IPP, VPPIPP, the preparation of the biochemical (Shanghai) Co., Ltd. of gill; Micropipet (100～1000 μ L, 20～200 μ L, 10～100 μ L, 0.5～10 μ L), Eppendorf Ltd; Strainer (Φ 50mm), the Shanghai institute of Pharmaceutical Industry; Millipore filtration (Φ 50mm, the aperture is 0.22 μ m), the Shanghai fast science equipment company limited that rubs; Centrifuge 5415 D small-sized high speed centrifugal machines, Eppendorf Ltd;

10 chromatographic systems, and Superdex 30 prep grade chromatography columns (GE Healthcare, USA); Zorbax SB-C18 reverse-phase chromatographic column (Agilent); The mole ultrapure water machine, Shanghai Moller scientific instrument company limited; The GL-22M high speed freezing centrifuge, Shanghai Lu Xiang instrument whizzer instrument factory; JY2002 type electronic balance, the flat instrument of last current chart company limited; Portable pressure steam sterilizer, Shanghai Medical Nuclear Instrument Factory; HWS26 type electric-heated thermostatic water bath, Shanghai one permanent Science and Technology Ltd.; Lab Dancer test tube vibrator, IKA Works Guangzhou; Electrolux BCD-252T type refrigerator, Elex (China) Appliances Co., Ltd.; DW-HW138 type Ultralow Temperature Freezer, middle section U.S. water chestnut low temperature science and technology limited Company; Analytical balance, Meitelei-tolido, German; Rat blood pressure meter: SoftronBP-98A, the soft grand Bioisystech Co., Ltd in Beijing.

The external ACE enzyme inhibition activity interpretation of result of synthetic six peptide VPPIPP:

Horse urea acyl histidyl-leucine (hippuryl-L-histidyl-L-leucine, HHL) under the catalysis of ACE enzyme, decompose generation urobenzoic acid (Hippuric Acid fast, HA) and dipeptides histidyl-leucine (HL), after adding the ACE enzyme inhibitors, the activity of ACE enzyme is suppressed, the growing amount of HA and HL reduces, by the RP-HPLC method (chromatographic column: Zorbax SB-C18 post (

5 μ m, 4.6mm * 250mm; Moving phase: 20% (v/v) analytical pure acetonitrile+0.1% (v/v) trifluoroacetic acid; Flow velocity: 1.0 ml/min)) growing amount of HA under the mensuration 228nm is estimated the ACE enzyme inhibitors to the inhibiting rate of ACE enzyme by following formula.

ACE enzyme inhibition rate (%)=100 * (A contrast-A sample)/(A contrast-A blank)

A contrast: 100mM sodium borate buffer liquid (BBS, the peak area of generation HA when pH8.3) replacing six peptides; A sample: the peak area that generates HA during six peptide samples; The A blank, hydrochloric acid adds fashionable 100mM sodium borate buffer liquid (BBS, the peak area of generation HA when pH8.3) replacing six peptides prior to HHL.Reaction system and condition see Table 1:

Table 1

ACE enzyme in the present embodiment extracts by the rabbit lung, through enzyme activity determination, prove that its enzyme activity is that a unit of enzyme of 0.732mU/mL((U) is defined as under this experiment condition, required enzyme amount when every min catalysis three peptide substrates (HHL) generate 1 μ mol product HA), satisfy requirement of experiment.So synthetic VPPIPP and two kinds of tripeptides Xie Ansuans (Val)-proline(Pro) (the Pro)-proline(Pro) (Pro) of being used widely (being called for short VPP), Isoleucine (Ile)-proline(Pro) (Pro)-proline(Pro) (Pro) (being called for short IPP) are tested, the results are shown in Table 2, as can be seen from the results, the relative size of the average inhibiting rate of the little peptide of hypertension when little peptide concentration is 10 μ M that record of experiment is IPP〉VPP ≈ VPPIPP.The above results shows that the ACE enzyme of polypeptide VPPIPP of the present invention has restraining effect, and then illustrates that also polypeptide VPPIPP of the present invention has the effect that brings high blood pressure down.

Table 2

Biological activity interpretation of result in the body of six peptide VPPIPP:

Six peptides in vivo with the ACE enzyme reaction before, also will be through digestion and the effect that absorbs, and physicochemical environment in vivo very complicated, perhaps accurate inadequately in external analog result of carrying out.Therefore we are by weighing the hypertension effect of six peptides to the variation of blood pressure behind spontaneous hypertensive rat (the Spontaneously Hypertensive Rat SHR) biologically active peptides of feeding.According to the characteristics of external activity data, selected positive control IPP and VPPIPP to carry out the interior biological activity confirmatory experiment of body, be rat administration classification in the table 3:

Table 3

Fig. 1,2 irritates blood pressure situation behind the stomach for rat (body weight is 400g), by test-results as can be seen, the SHR Hypertensive Rats at feeding behind two kinds of antihypertensive active peptides, decline has all taken place in blood pressure.Rat is fed the IPP(1.5mg/kg body weight) after, the blood pressure of rat the trough that significantly decreases, blood pressure is initially the normal hypertension level about 195, descend beginning after about 1 hour behind the feeding IPP, after the administration about 3 hours, drop to minimumly 160～165, administration has returned to about 195～200 normal hypertension level again after 6 hours, as shown in Figure 1.Rat is fed the VPPIPP(1.5mg/kg body weight) after, tangible trough has also appearred in pressure value, be approximately 4 hours the working lipe of medicine, blood pressure begins to descend behind 0.5h, drop to about 150 from about 200 hypertension level, lower-most point appeared at administration after 2 hours, and administration returns to 200 initial levels again after 4 hours, as shown in Figure 2.Blank group rat has been fed behind the 4mL deionized water, though the data that obtain are not ideals very, the standard deviation of data is bigger, can obviously judge, and blood pressure is significantly reduction not, as shown in Figure 3.The above results shows that the present invention six peptide VPPIPP have the better blood pressure lowering effect than IPP in the test in vivo.

Claims (3)

1. antihypertensive active peptide, it is characterized in that: the aminoacid sequence of this bioactive peptide is: VAL-PRO-PRO-ILE-PRO-PRO.

2. antihypertensive active peptide as claimed in claim 1 is in preparation prevention or treat application in hypertensive medicine or the food.

3. an antihypertensive composition wherein contains the bioactive peptide described in the claim 1 as effective constituent, and contains conventional pharmaceutical carrier and/or vehicle.

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