CN102276695B - Polypeptide with function of lowering blood pressure and application thereof - Google Patents

Abstract

The invention belongs to the field of biological pharmacy and in particular relates to polypeptide with the function of lowering blood pressure and application thereof. The invention aims to solve the technical problem of providing new blood pressure lowering polypeptide. The technical scheme for solving the technical problem is as follows: the polypeptide with the function of lowering blood pressure is provided. The polypeptide with the function of lowering blood pressure is (1) the polypeptide with the amino acid sequence in the SEQ ID No.3 or (2) the polypeptide with the same or similar function after one or more amino acids are replaced, deleted or added on the basis of the amino acid sequence in the SEQ ID No.3. In vivo and in vitro experiments prove that the polypeptide has good function of lowering blood pressure, can be prepared into blood pressure lowering drugs and provides a new effective selection for the blood pressure lowering field.

Description

Has polypeptide of antihypertensive function and uses thereof

Technical field

The invention belongs to field of biological pharmacy, be specifically related to a kind of polypeptide with antihypertensive function and uses thereof.

Background technology

Pig blood has long medicinal history, and from the Han dynasty, the successive dynasties book on Chinese herbal medicine is all listed pig blood separately, has put down in writing its medicinal efficacy: " replenishing blood for nourishing heart, it is relieving convulsion to relieve dizziness, high fever, infantile convulsions, epilepsy, etc., the therapeutic method to keep the adverse qi flowing downward, hemostasis " etc.; Also included a large amount of proved recipes simultaneously.Also extensively edible pork blood prevention among the people, treatment disease.Modern study also shows that pig blood not only contains necessary 8 seed amino acids of human body at 18 interior seed amino acids, has higher nutritive value, can also treat anaemia, leukemia, and blood fat reducing promotes wound healing etc.Yet people have mainly utilized the consumption of this kind, have ignored its medicinal efficacy to a great extent now.This mainly is because pig blood medicinal function basic substance is not clear, and is also very weak to its medicinal basic research.

Abroad particularly Japan is raw material with pig blood, behind the research enzymolysis biologically active peptides, the partial function of polypeptide is put down in writing with book on Chinese herbal medicine and is conformed to.China's pig blood resource is abundant, and annual production is about 1,000,000,000 kilograms, but only has sub-fraction to be applied to food, feed industry, and the overwhelming majority becomes waste.With China historical background similarly Japan be about 50～60% (mainly record utilizes modern biotechnology to carry out being developed as medicine after the deep processing according to book on Chinese herbal medicine) to the utilization rate of pig blood, it mainly is domestic not enough to this resource attention degree tracing it to its cause, effective constituent is indeterminate, is difficult to degree of depth utilization.Rich and the low-level history of present situation and book on Chinese herbal medicine record use and the temperature of foreign study utilization now utilized of domestic this resource impels us to draw attention to this resource.

Abroad the bioactive peptide that is obtained by protein digestion is one of functional foodstuff, drug development research most active fields.The investigator has found some small-molecular peptides from the zymolyte of food protein, these peptides mainly can be divided into by physiological function: the peptide of the phosphopeptide caseinate of morphine sample bioactive peptide, antihypertensive peptide, immunomodulatory peptides, promotion calcium absorption, antithrombotic peptide, the peptide that promotes the synthetic and cell growth of DNA, antibacterial peptide, anticancer peptide, promotion bifidobacterium growth etc.

Be that the biologically active peptides of protein source is by behind the modern biotechnology enzymolysis oxyphorase in the pig blood being converted into small-molecular peptides with pig blood.Studies show that, small molecules pig hemepeptide not only has good solvability, low viscosity, current research shows, oligopeptides in the diet (2～3 amino acid) and polypeptide (10～51 amino acid) can intactly pass through intestinal absorption, are organizing level to cause the biological effect of body as biologically active peptides.And, the protein utilization height, it also has low antigenicity, can not produce anaphylaxis.

Dardenne in 1977 separates from porcine blood serum with Pleau etc. and has made with extra care immunomodulatory hormone-Zadaxin (TF), and finishes the structure analysis, has obtained its amino acid, molecular weight 857Da.The great grade of China Feng Qi shows that by pharmacological research the TF that obtains has significant immunocompetence from pig blood.It is the functional polypeptide in source that increasing research is paid close attention to the porcine haemoglobin.People's enzymolysis pig blood globins such as Kazue Mito have obtained 4 kinds and have had the polypeptide that suppresses the ACE activity.Yanmei ZHANGs etc. carry out separation and purification and anti-microbial activity research to antibiotic peptide matters in the pig blood; Wang Yanzhuo separates from pig blood and obtains the polypeptide that molecular weight is 6kDa, and it is best to intestinal bacteria Quality Control strain ATCC25922 anti-microbial activity.Fang Jun etc. study porcine haemoglobin polypeptide anti-oxidant function.Need in pig blood, find new active polypeptide at present, think that this area provides new selection.

Summary of the invention

Technical problem to be solved by this invention provides new functional polypeptide.

The technical scheme of technical solution problem of the present invention provides a kind of new polypeptide with antihypertensive function.This has the polypeptide of antihypertensive function:

(1) is the polypeptide with the described aminoacid sequence of SEQ ID No.3;

Perhaps be:

(2) be the aminoacid sequence basis shown in the described SEQ ID No.3 through replacement, disappearance or add one or several amino acid after and polypeptide with same or similar function.

The function of aforementioned polypeptides is hypotensive.

The present invention also provides above-mentioned coding nucleotide sequence with polypeptide of antihypertensive function.

The present invention also provides the carrier that contains above-mentioned coding nucleotide sequence.

The present invention also provides the host cell that contains above-mentioned carrier.

The present invention also provides the above-mentioned purposes of polypeptide in the preparation Altace Ramipril with antihypertensive function in addition.

Certainly, the present invention also provides a kind of new Altace Ramipril.This Altace Ramipril contains the above-mentioned polypeptide with antihypertensive function as effective constituent.

Further, the formulation of above-mentioned Altace Ramipril is oral preparations.

Further, above-mentioned oral preparations is mainly capsule, tablet, dripping pill, lozenge, dispersible tablet, tablet or granule etc.

The present invention is initial research material with pig blood.At first make pig blood destainer, then pig blood destainer is carried out further enzymolysis.After the enzyme class compared, it was good still selecting for use stomach en-to carry out enzymolysis at last, and by the stomach en-enzymolysis, ultrafiltration then obtains the enzymolysis solution MC of molecular weight＜3kDa.

Subsequently again with enzymolysis solution MC by gel chromatography separate, oppositely high performance liquid chromatography is separated, external ACE suppresses active the tracking and obtain 6 novel polypeptides, Genebank polypeptide data base querying shows that these 6 kinds of peptides are to separate first and obtains from pig blood.And by ground substance assistant laser desorption ionization flight time mass spectrum (MALDI-TOF-MS) mensuration sequence, wherein a kind of aminoacid sequence is VVYPW, and it is to the active IC that suppresses of ACE ₅₀Be 0.254mg/ml.

The VVYPW polypeptide has been carried out stomach en-and tryptic digestion experiment, digested the back increased activity, illustrated that this peptide can the oral administration administration.Infer that the active inhibiting peptide of this ACE is precursor type inhibitor (Prodrug-type inhibitor).Pharmacological evaluation shows that this polypeptide has significant hypotensive activity to spontaneous hypertensive rat (SHR).Can bring into play drug effect in 1～36 hour after administration, hypotensive activity in 3 hours is the strongest after the administration.Just confirmed its blood pressure lowering effect.

This polypeptide can be used as other complementary compositions that activeconstituents adds this area again, or adds other compositions with anti-blood pressure activity again and make the preparation use.Its formulation is preferably oral preparations, as: common oral preparations such as capsule, tablet, dripping pill, lozenge, dispersible tablet, oral liquid, tablet or granule.

The present invention has obtained to have the polypeptide VVYPW (SEQ ID No 3) of good antihypertensive activity first, and prove that by internal and external test its synthetic peptide has good hypotensive activity, can prepare becomes Altace Ramipril, and can orally use, for hypotensive field provides a kind of new effective selection, have good market outlook.

Description of drawings

Fig. 1 pig blood destainer SDS-PAGE (Tris-Glycine system) electrophoretogram.M: standard substance 1: stomach en-enzymolysis solution 2: trypsin digestion liquid 3:1394 protease hydrolyzed liquid 4: papain enzymolysis liquid.

Fig. 2 pig blood destainer SDS-PAGE (Tris-Tricine system) electrophoretogram.M: standard substance 1: stomach en-enzymolysis solution 2: after the ultrafiltration of stomach en-enzymolysis solution 3: trypsin digestion liquid 4: after the ultrafiltration of trypsin digestion liquid molecular weight 5: papain enzymolysis liquid 6: after the ultrafiltration of papain enzymolysis liquid after the ultrafiltration of 7:1394 protease hydrolyzed liquid 8:1394 protease hydrolyzed liquid molecular weight

Fig. 3 Sephadex G-25 gel chromatography separation collection of illustrative plates.I, II, III, IV, V, VI represent component (pipe number) I, II, III, IV, V, VI respectively.

The SHRs single administration blood pressure figure of Fig. 4 P1, P2, P3.SHRs single administration SBP (systolic pressure) changes as mentioned above, and vertical bar represents standard deviation.Wherein: ● represent blank group (pure water), △ represents the P1 group, and ◇ represents the P2 group, and represents the P3 group.Ordinate zou is represented rat caudal artery systolic pressure changing value, and X-coordinate is represented the time after the administration.Compare with control group, significance is *, p＜0.05.

SHRs single administration blood pressure (SBP, the systolic pressure) change curve of Fig. 5 P4.Wherein: ● represent blank group (pure water), ■ represents the P4 group.Ordinate zou is represented rat caudal artery systolic pressure changing value, and X-coordinate is represented the time after the administration.Compare with control group, significance is *, p＜0.05.

SHRs single administration blood pressure (SBP, the systolic pressure) change curve of Fig. 6 P5.Wherein: ▲ representing blank group (pure water), ■ represents the P5 group.Ordinate zou is represented rat caudal artery systolic pressure changing value, and X-coordinate is represented the time after the administration.Compare with control group, significance is *, p＜0.05.

SHRs single administration blood pressure (SBP, the systolic pressure) change curve of Fig. 7 P6.▲ representing blank group (pure water), ■ represents the P6 group.Ordinate zou is represented rat caudal artery systolic pressure changing value, and X-coordinate is represented the time after the administration.Compare with control group, significance is *, p＜0.05.

Embodiment

Be further described by the inventive method of embodiment below in conjunction with accompanying drawing, but therefore the scope of protection of the invention be not limited among the scope of embodiment.

The external enzymolysis of pig blood of embodiment one analogue body intracellular metabolite

The dried main component of pig blood is protein, and content is nearly 20%, and wherein the oxyphorase in the hemocyte accounts for 75% of whole blood Tot Prot.Oxyphorase is degraded into the small-molecular peptides compounds of different chain length after hydrolysis.Colour developing is mainly protoheme with stench flavor material in the pig blood, and research is serious disturbs to the extraction separation of active substance, assay, molecular weight detection etc. for it.The human body of going into of pig blood at first is degraded into more stable polypeptide by digestive ferment (molecular weight ranges is that 0.1kDa～10kDa), molecular weight is less than the common biologically active of little peptide of 3kDa, and the little peptide of this class also easily is absorbed by the body.The little peptide of this class also is the general character material of pig blood onset.Therefore select suitable method the decolouring of pig blood to be become the top priority of seeking pig blood antihypertensive activity composition.

In conjunction with activated carbon decolorizing, the decolouring product adopts digestion condition in the in-vitro simulated body of digestive ferment, destainer is carried out further enzymolysis, and enzymolysis solution is carried out ultrafiltration, obtains molecular weight less than the polypeptide crude product (MC) of 3kDa by enzymolysis in the present invention.Whole process is followed the tracks of with the molecular weight ranges of the albumen of Tricine-SDS-PAGE, polypeptide.For further separation and purification step-down peptide lays the foundation.

1, experiment material and instrument

Pig blood (being collected in the slaughterhouse, Wenjiang); Stomach en-, trypsinase, 1394 enzymes, papoid, the biochemical factory in Deyang; Powdered Activated Carbon (the lucky first chemical company limited in Chongqing); Cupric sulfate pentahydrate, anhydrous sodium carbonate, trisodium citrate, acetic acid, glycerine, sodium hydroxide, sodium starch glycolate, the Long Huagongshijichang of Chengdu section; The forint phenol reagent; Sodium lauryl sulphate; Acrylamide (acrylamide), N, N-Methylene bisacrylamide (methylene diacrylamide), Tris (Tutofusin tris), Tricine (N-three (methylol) methylglycine), Glycin (glycine), U.S. sigma company; More than be all analytical pure.

Ultraviolet-visible pectrophotometer (France Anthelie); Refrigerated centrifuge (Biofugo primor); PH meter (DD-15); Vortex mixed instrument (WH-3); Thermostat water bath; 3kD, 10kD Millipore ultrafiltration concentrator bowl; Electrophoresis apparatus (Mini-proteanIII); Ice-making machine (SIM-F124); Refrigerated centrifuge (Biofuge primor); Gel electrophoresis analysis system (170-8101)

2, pig blood decolouring research

2.1 experiment flow

Fresh pig blood → adding trisodium citrate anti-freezing → centrifugal → take off layer → enzymolysis → accent pH=3.0 → add sorbent material decolouring → centrifugal → get supernatant liquor → supply same volume → comprehensive evaluation decolorizing effect

2.2 experimental technique

2.2.1 protoheme is measured

With ultraviolet spectrophotometer destainer is carried out full wavelength scanner, obtain two maximum absorption band OD ₂₈₀And OD ₄₁₅, OD ₂₈₀Be the characteristic peak of protein, OD ₄₁₅Characteristic peak for protoheme.

OD is selected in this experiment for use ₄₁₅Carry out the mensuration of protoheme.Percent of decolourization calculates according to following formula:

The decolouring of As-enzymolysis solution is preceding at OD ₄₁₅Absorbance

A ₀-enzymolysis solution decolouring back is at OD ₄₁₅Absorbance

2.2.2 protein content determination

Adopt the lowry method ^[53]Measure the protein concentration of destainer sample, pig blood former state, calculate protein yield (PR).Method of calculation as shown in the formula:

2.2.3 response value is calculated

Decolorizing effect adopts the response value evaluation, has both guaranteed higher percent of decolourization, guarantees higher protein yield again, and response value is the smaller the better, method of calculation as shown in the formula:

2.2.4 enzyme activity determination

Adopt the method for the specialized standard ZBX66030-87 of the People's Republic of China (PRC) " protease activity amylograph " regulation.

2.2.5 decolorization condition optimization

Adopt 4 factors, 5 horizontal uniform designs to carry out decolorization condition optimization.

2.3 interpretation of result

2.3.1 discoloring agent is selected

Choosing Powdered Activated Carbon, sodium starch glycolate (CMS), Xylo-Mucine (CMC) is discoloring agent, evaluation index is protein yield, percent of decolourization, response value, color and luster, smell, remove difficulty or ease (investigating with the centrifugal time of removing), considers discoloring agent cost, regeneration problem.Use the stomach en-enzymolysis solution, decolorization condition is sorbent material add-on 4%, pH=3.0,55 ℃ of temperature, 30min, 8000g are centrifugal in reaction.Decolouring evaluation sees Table 1.

The different bleaching agent bleaching effect assessments of table 1

Discoloring agent	The color and luster degree	Smell	Response value R	Centrifugation time (min)
					CMS	++ +++pale brown	Light	?640	?35
Gac	++ pale yellow	Light	?123	?20

Annotate: "+" represents the color and luster degree, and more many representative colors are more dark, and "+" all represents this meaning in the following form, do not do repeat specification.

In experiment, because the CMC dissolution rate is very slow, almost can not dissolve in the reaction times, not be suitable for decolouring, abandon using.

By the decolorizing effect of response value comprehensive evaluation CMS and gac, gac slightly is better than CMS, and easily centrifugal removing considers that again the gac cost is lower, is discoloring agent so gac is all selected in following experiment for use.

2.3.2 decolouring enzymolysis enzyme class is selected

Choose stomach en-, trypsinase, 1394 enzymes, four kinds of enzyme enzymolysis of papoid porcine haemoglobin, enzymatic hydrolysis condition is that 6.0% enzyme concentration, 12.0% concentration of substrate, 3h action time, 40 ℃ of temperature, pH are each enzyme optimum pH (seeing Table 2), discoloring agent is gac, adopt single factor analysis, relatively decolorizing effect sees Table 3.

The suitableeest action condition of the various enzymes of table 2

The enzyme class	The pH value	Enzyme activity U
			1394 enzymes	7.2	131200
Trypsinase	7.2	40400
			Stomach en-	2.5	94600
Papoid	8.0	140000

The different enzyme class of table 3 decolorizing effect is estimated

The enzyme class	The color and luster degree	The smell degree	Response value R
				1394 enzymes	++	Light	34887
Trypsinase	+	Do not have	7232
				Stomach en-	++++	Light	1317
Papoid	+++	Dense	1867

As can be known from Table 3, except papoid, the flavor of all smelling as of rotten fish; Behind the stomach en-enzymolysis, the destainer color and luster is dark than other enzymes, but overall color is all superficial, and difference is little, is light yellow; And stomach en-destainer protein content height, by response value R comprehensive evaluation, decolorizing effect obviously is better than other three kinds of enzymes.Stomach en-is main digestive ferment, and it plays a role under about sour environment pH=3.0, this pH value down sphaeroprotein be combined with protoheme the most loose, so select for use stomach en-for carrying out enzymolysis.

2.3.3 the stomach en-decolorization condition is optimized

The stomach en-decolorization condition is optimized, chooses pH=3,55 ℃ of temperature, 4% discoloring agent add-on, activated carbon decolorizing.Temperature, time, enzyme concentration, 4 indexs of concentration of substrate are set, and 5 levels (seeing Table 4) are carried out homogeneous design (seeing Table 5).

Table 4 influences 4 factors, the 5 levels design of hydrolysis result

Table 5 optimization Test scheme and result

The color and luster degree that No. 1～5, sample, smell degree are distinguished little, and sense organ can not judge that color and luster is faint yellow, the stench flavor of nothing on the whole.

Be Uniform Design result (seeing Table 5) with response value R, carry out statistical procedures:

P＜0.1 can be selected in R=0.999 F=190.706 P=0.053＜0.1 in real work.

Each number of levels of evenly showing is changed to concrete level value, and calculating progressively, regression equation gets:

Y＝5454.517-65.457x2-166.771x4-437.4x1

Every by regression equation is negative sign, and as can be seen, so the approximate evaluation of optimum point is x1=2.0 x2=45 x3=0.5x4=14.4, the decolouring preferred embodiment is: enzyme concentration 8.0%, 45 ℃ of temperature, enzymolysis time 0.5h, concentration of substrate 18.0%.

3, external enzymolysis analogue body intracellular metabolite

3.1 experiment flow

Destainer → enzymolysis → high temperature enzyme → centrifugal (4000g * 40min) → get supernatant liquor → successively carry out ultrafiltration with the millipore filter membrane of 10kDa, 3kDa that goes out

With SDS-PAGE electrophoresis (Tris-Tricine, Tris-Glycine system), two kinds of methods of lowry method are carried out molecular weight ranges respectively and are followed the trail of and content detection.

3.2 method

3.2.1 ultra-filtration and separation

For removing macromolecular interference, obtain the peptide of molecular weight＜3kDa, select the filter membrane of 10kDa and 3kDa to carry out the segmentation ultrafiltration.Get each 10ml of enzymolysis solution behind the enzyme that goes out, carry out ultrafiltration with the filter membrane of 10kDa earlier and remove macromolecular interference, follow the filtrate of molecular weight＜10kDa is carried out ultrafiltration with the filter membrane of 3kDa, obtain the enzymolysis solution MC of molecular weight＜3kDa, respectively recording volume and keeping sample.

3.2.2 determining content of peptides

Adopt the lowry method to measure the content of peptides of the ultrafiltrated of go out enzyme enzymolysis solution, polypeptide molecular weight＞3kDa, calculate molecular weight＜3kDa polypeptide yield PR value, as main evaluation index, compare the hydrolysis result of each enzyme with the PR value.Method of calculation as shown in the formula:

PR＝[(W1-W2)/W1]×100％

W1---the content of peptides of the enzyme enzymolysis solution that goes out

The content of peptides of W2---molecular weight＞3kDa/10kDa ultrafiltrated

3.2.3 the peptide molecule weight range is measured

This experiment with the polypeptide of molecular weight＜3kDa as the target peptide group, so adopt conventional SDS-PAGE, Tris-Glycine electrophoresis system ultra-low molecular amount SDS-PAGE, the Tris-Trcine electrophoresis system detects analysis to the polypeptide molecular weight of enzymolysis solution.Deposition condition sees Table 6

Table 6 Tris-Glycine Tris-Trcine system deposition condition

3.3 interpretation of result

3.3.1 the peptide molecule weight range is measured

Choose the enzymolysis solution of stomach en-, trypsinase, 1394 enzymes, four kinds of enzymes of papoid, carry out SDS-PAGE (Tris-Glycine system) electrophoresis, the enzymolysis solution electrophoretogram is not seen band as shown in Figure 1, show enzymolysis after molecular weight analyte all less than 14.4kDa, illustrate that enzymolysis all compares thoroughly.

Sample carries out SDS-PAGE Tris-Tricine system electrophoresis after choosing stomach en-, trypsinase, 1394 enzymes, four kinds of enzyme enzymolysis solutions of papoid and 3kDa ultrafiltration, polypeptide molecular weight distributes all below 14.4kDa in each protease hydrolyzed liquid as shown in Figure 2, and it is more thorough to further specify enzymolysis.After the 3kDa ultrafiltration, molecular weight distribution is all below 3.3kDa.And, all deep for band color before and after the ultrafiltration of protease hydrolyzed liquid, illustrate that its peptide concentration is higher.

3.3.2 content of peptides analysis

Choose stomach en-, trypsinase, 1394 enzymes, four kinds of enzyme enzymolysis of papoid destainer, enzymatic hydrolysis condition is that 6% enzyme concentration, 12% concentration of substrate, 6 hour action time, 40 ℃ of temperature, pH are each enzyme optimum pH (seeing Table 2), use less than the main evaluation index of 3kDa polypeptide yield conduct, comprehensively compare the hydrolysis result of each enzyme.(seeing Table 7)

The different enzymatic hydrolysis conditions of table 7＜3kDa polypeptide yield

The enzyme class	Polypeptide total content (mg)	Polypeptide yield (%)
			Stomach en-	1982.37	?88.86
Trypsinase	802.56	?98.56
			Papoid	816.0	?66.21
1394 enzymes	561.79	?93.21

Behind stomach en-, trypsinase, 1394 enzyme enzymolysis, the polypeptide yield of molecular weight＜3kDa is all higher as shown in Table 7, and the polypeptide yield of papoid molecular weight＜3kDa is relatively low.The polypeptide total content of trypsinase, papoid, 1394 enzymes is less, and pepsin content is higher, illustrates that pepsic hydrolysis result is relatively good.Papoid is plant protease, the 1394th, and microbial protease, pig blood plays a role in human body, needs to decompose through digestive enzyme, and stomach en-is main digestive ferment in the human body, comprehensive evaluation, stomach en-is the suitableeest proteolytic enzyme.

By experiment of single factor, it is relatively good to investigate out the stomach en-decolorizing effect, and its decolorization condition is enzyme concentration 8.0%, 45 ℃ of temperature, enzymolysis time 0.5h, concentration of substrate 18.0%, and sorbent material is gac.The destainer that obtains with this understanding yellow of light color, no fishy smell, protein content is higher.Remove substance that show color (stench material), can effectively reduce the influence of experiments such as its extraction separation to functional polypeptide, assay.The further enzymolysis of pig blood destainer compares the enzyme class, and comprehensive evaluation still selects for use stomach en-to carry out enzymolysis at last, the polypeptide total content that obtains molecular weight＜3kDa by the stomach en-enzymolysis is the highest, reach 20%, and the polypeptide yield height of molecular weight＜3kDa, reach 88.86%.

The present invention selects the digestive enzyme stomach en-to carry out enzymolysis decolouring, has not only simplified the decolouring step, obtains good decolorizing effect, has guaranteed the yield of polypeptide, and laid the foundation for next step utilizes digestive enzyme analogue body intracellular metabolite.

Embodiment two pig blood antihypertensive function polypeptide separate and identify

Pathogenesis of hypertension is commonly referred to be hypertensinase (ACE) makes angiotensin I (Ang I) excessively convert the Angiotensin II (AngII) that makes elevation of blood pressure to, and the bradykinin of ACE degraded simultaneously makes it inactivation.So reduce the generation of Angiotensin II by the activity that suppresses ACE, the degraded of impedance bradykinin (inactivation) thus bring high blood pressure down.Cushman proposes ACE the earliest in the rabbit lung, set up ACE and suppressed active measuring method, and many scientific research personnel changed on this basis afterwards.This experiment filters out pig blood antihypertensive function polypeptide by the interior restraining effect to ACE of in-vitro simulated body.

After this experiment separates by gel-filtration, adopt the smart separation of RP-HPLC, the active tracking of sepn process employing ACE, the polypeptide that obtains adopts ground substance assistant laser desorption ionization flight time matter (MALDI-TOF-MS) mensuration sequence, and reliable results is accurate.Obtaining antihypertensive function polypeptide and the aminoacid sequence thereof of pig hemase solution, and the essential property of polypeptide has been carried out preliminary research.

1, experiment material and instrument

Molecular weight 3kDa enzymolysis solution MC; HHL, ACE, TNBS (Sigma Chemical Co.); Captopril (Chengdu medicine inspecting institute); Glacial acetic acid (analytical pure), sodium acetate trihydrate (analytical pure), acetonitrile (chromatographically pure), S-WAT, boric acid, Sodium Tetraborate, methyl alcohol (chromatographically pure) dextrane gel Sephadex G-25 (Amersham Biosciences).

Spontaneous hypertensive rat (SHR) (Shanghai Si Laike animal center), SD rat (Chengdu University of Traditional Chinese Medicine's Experimental Animal Center).

Ultraviolet-visible pectrophotometer (France Anthelie); Refrigerated centrifuge (Biofugo primor); PH meter (DD-15); Vortex mixed instrument (WH-3); Thermostat water bath; Peristaltic pump; The substep collector; Microplate reader (Varioskan); Ice-making machine (SIM-F124); High performance liquid chromatograph (LC-20A, day island proper Tianjin company); Chromatographic column ((the Shim-pack VP-ODSC of 4.6mm * 150mm) ₁₈Column).

2, the separation of active antihypertensive peptide

2.1 the chromatographic separation of active antihypertensive peptide

2.1.1 gel chromatography column condition

The dress post: gel chromatography column 1.6cm * 60cm, get 10g Sephadex G-25 filler, add distilled water stirring heating swelling 1.5h, take out and be cooled to room temperature, add the 0.5mol/LNaOH-0.5mol/LNacl pre-treatment, suction filtration cleans up repeatedly with distilled water; Wet method dress post;

Pre-equilibration: with moving phase pre-equilibration 30min, flow velocity 1.5ml/min;

Post imitate to detect: get sample on the acetone of 1% volume, flow velocity 1.5ml/min detects wavelength 280nm.

2.1.2 sample initial gross separation

Get 2ml (3.3%v/v) molecular weight and go up sample less than 3kDa enzymolysis solution MC, (0.05mol/L pH=5.0) carries out wash-out, detects wavelength 215nm, and substep is collected with acetic acid-sodium acetate.

2.2 the RP-HPLC of active antihypertensive peptide separates

Get the strongest component of activity of initial gross separation, with the water membrane filtration of 0.45 μ m, last sample.

High-efficient liquid phase technique separates 37 ℃ of blood pressure lowering polypeptide column temperatures, detects wavelength 215nm, adopts water-acetonitrile gradient wash-out to separate mobile phase A: water (containing 5% acetonitrile); Mobile phase B: acetonitrile (containing 5% water); Gradient elution: 1%-100%B 60 minutes.

2.3 hypotensive activity detects

With reference to Matsui T (Matsui T, Matsufuji H, Osajima Y.Colorimetric measurement of angiotensin I-converting enzyme inhibitory activity with trinitrobenzene sulfonate.Biosci Biotechnol Biochem.1992,56 (3): 517-518.) and the method for US 2007/0141120A1-0084 patent documentation record, and suitably change.Be specially: take the 5mlEP pipe, add testing sample by table, place 37 ℃ of insulation 20min, add corresponding reagent again, place 37 ℃ of insulation 20min again, measure optical density A under the 416nm, calculate the ACE inhibiting rate, the inhibitor concentration that the ACE inhibiting rate is reached at 50% o'clock is 503nhibiting concentration, counts IC ₅₀Operation steps such as following table 8.

Table 8 ACEI determination of activity application of sample table

Blank1 is the blank of Control, and Blank2 is the blank of Sample, is used for measuring inhibiting rate:

$\mathrm{IR}\left(\mathrm{Inhibitoryratio}\right)=\frac{(\mathrm{Ac}-\mathrm{Ab}1)-(\mathrm{As}-\mathrm{Ab}2)}{(\mathrm{Ac}-\mathrm{Ab}1)}\×100\%$

The enzyme unit definition (U) of living: the enzyme work of 1 unit is defined as the enzyme amount that in 37 ℃, 1min catalysis HHL forms the 1umol urobenzoic acid.

2.4 interpretation of result

The MC that ultrafiltration obtains obtains a series of active ingredients by the gel chromatography initial gross separation, sees Table 9; What wherein activity was the strongest is component I, and inhibiting rate reaches 100%; Component I obtains 6 polypeptide P1, P2, P3, P4, P5, P6 by the RP-HPLC separation, sees Fig. 3.They have ACE to suppress active, see Table 10.

Table 9 active ingredient

The component sequence number	With volume inhibiting rate %
		Component I	100％
Component I I	61％
		Component III
	40％
		Component I V
	24％
		Component V	33％
Component VI	19％

Table 10 separates the purifying table of six ACE active polypeptide that obtain

Title	Sequence	Molecular weight (Da)	The rate of recovery (%)	The purifying multiple	IC 50mg/ml
						Captopril	/	/	/	/	4.371
MC	/	/	/	/	0.397
						P1(SEQ?ID?No?1)	WVPSV	586.69	0.11	166	0.368
P2(SEQ?ID?No?2)	YTVF	528.61	0.13	125	0.226
						P3(SEQ?ID?No?3)	VVYPW	662.79	0.08	182	0.254
P4(SEQ?ID?No?4)	VTSF	659.74	0.12	148	0.228
						P5(SEQ?ID?No?5)	EGQLTL	713.87	0.16	108	0.525
P6(SEQ?ID?No?6)	WVPSVY	749.86	0.19	95	0.425

3, the evaluation of active antihypertensive peptide

3.1 active antihypertensive peptide mass spectroscopy

Liquid-phase condition:

NanoACQUITY UPLC Ultra Performance Liquid Chromatography system disposition automatic sampler.

Enriching column: Symmetry C18,180 μ mx20mm, 5um particle.Analytical column: the nanoACQUITYUPLC Ultra Performance Liquid Chromatography bonding ethane particle post BEH C that mixes ₁₈, 75um * 250mm, 1.7um particle.Column temperature: 35 ℃; Flow velocity: 200nl/min.Mobile phase A: contain the aqueous solution of 0.1% formic acid, Mobile phase B: the second cyanogen that contains 0.1% formic acid.Gradient: 1% arrives 40%B 65 minutes, arrives 85%B 5 minutes, and 80%B stopped 10 minutes, and balance arrived initial 1%B in 10 minutes.

The mass spectrum condition

Mass spectrograph: Synapt High Definition Mass Spectrometry mass spectrograph (Waters company).Source temperature: 90 ℃.Acquisition range: MS:350-1600Da, MS/MS:50-2000Da.

Data analysis software: PLGS v2.3 handles the back by the MS MS ionic means searching database of Mascot retrieval software.Search condition: the Trypsin enzyme is cut, and M oxidation and iodo-acid amide alkyl turn to variable modification, and it is 1 that the site is cut in leakage.The quality error of MS and MS/MS is 0.2Da.

3.2Genebank the polypeptide database is investigated

6 polypeptide that the mass spectrum order-checking obtains finally show all to be difficult the polypeptide log-on message at Genebank polypeptide data base querying (http://www.ncbi.nlm.nih.gov/peptidome/resources/), tentatively are indicated as acquisition first.

3.3 active antihypertensive peptide is synthetic

Polypeptide is synthetic by Shanghai Bootech BioScience ﹠amp; Technology Co., Ltd. company is synthetic.

3.4 interpretation of result

Six polypeptide in the component I are identified, obtained aminoacid sequence, be respectively: WVPSV, VVYPW, YTVF, WVPSVY, EGQLTL, VVLLGDV.Their molecular weight all below 1000Da, maximum 749Da, minimum 528Da.P1, P2, P3, P4, P5, P6 are the active inhibiting peptide of the ACE that separates first from pig blood, wherein, P3 (VVYPW) is than active inhibiting peptide VVYPWY (SEQ ID the No.7) (IC of the ACE of former bibliographical information ₅₀Be 0.62mg/ml) few amino acid Y, IC ₅₀Also obvious little (IC ₅₀The active inhibit feature of more little its ACE is more strong).

4, active antihypertensive peptide study on the stability

4.1 thermostability is investigated

Because polypeptide source enzymolysis solution MC obtains by high temperature (T=90 ℃) enzyme that goes out, so these polypeptide have thermostability preferably.

4.2 the digestion enzyme stability is investigated

Get the digestion experiment that trypsinase and stomach en-carry out blood pressure lowering peptide, investigate it to the stability of gastrointestinal enzyme.Sample before and after the enzymolysis has carried out ACE and has suppressed determination of activity.Get the 1mg synthetic peptide respectively, respectively get the stomach en-of a 100 μ l 3% of adding, another part adds the trypsin solution mixing of 100 μ l 3%.At 37 ℃ of reaction 6h, take out centrifugal (8000g * 10min), get supernatant liquor and measure ACE inhibition activity.

4.3 interpretation of result

All there is ACE to suppress active before P1, P2, P3, P4, P5, the P6 digestion.Activity is not high before P1, the P2 digestion, IC ₅₀Value is respectively 5.2%, 6.4%.P1 behind gastric pepsin digestion, increased activity, IC ₅₀Value 0.336mg/ml; P2 behind tryptic digestion, increased activity, IC ₅₀Value 0.210mg/ml.P3 behind stomach, tryptic digestion, increased activity, IC ₅₀Value is: 0.248mg/ml.P4 by gastric pepsin digestion after, increased activity, IC ₅₀The value 0.220mg/ml, P5 by gastric pepsin digestion after, increased activity, IC ₅₀The value 0.520mg/ml, P6 by gastric pepsin digestion after, increased activity, IC ₅₀Value 0.415mg/ml.

Show that tentatively P1, P2, these three kinds of peptides of P3 are difficult for being degraded and losing activity by stomach en-, can be applicable to oral administration.They are being produced the more active small peptide fragment of small molecular weight by the increased activity explanation of digestion back after digesting, this just is more conducive to the absorption of intestinal mucosa cells in the body and the performance of effect.The exclusive digestive ferment of P1 may be stomach en-, and the exclusive digestive ferment of P2 may be trypsinase, and P3 has affinity to two kinds of enzymes.

The checking of embodiment three blood pressure lowering peptide pharmacology

Confirm to have ACE through experiment in vitro and suppress active blood pressure lowering peptide, might not all have hypotensive effect in vivo.Blood pressure lowering peptide must enter its hypotensive effect of blood circulation competence exertion, if oral, by the decomposition of the enzyme in the digestive tube or by ACE degraded in advance, all may become small peptide or the amino acid of non-activity easily.Can the blood pressure lowering peptide that the present invention obtains successfully pass through GI barrier? by experiment in the body as experimentation on animals or clinical trial, effect that can more accurate judgement blood pressure lowering peptide.

In the experiment in front, obtained to have ACE and suppressed 6 active blood pressure lowering peptides.In order further to verify the actual antihypertensive effect of these blood pressure lowering peptides, this experimental selection P1, P2, P3, P4, P5, P6 have carried out short-term administration experiment to spontaneous hypertensive rat (SHR), the buck functionality of research blood pressure lowering peptide.

1, experiment material and instrument

Synthetic polypeptide P1 (SEQ ID No 1), P2 (SEQ ID No 2), P3 (SEQ ID No 3), P4 (SEQ ID No4), P5 (SEQ ID No 5), P6 (SEQ ID No 6); Salt-free or less salt mouse grain (Chengdu University of Traditional Chinese Medicine experimentation on animals center);

Male spontaneously hypertensive rat (SHR);

BP-6 animal non-invasive blood pressure tester (Chengdu TME Technology Co., Ltd.).

2, method

2.1 laboratory animal pre-treatment

According to the method that Michio Muguruma introduces, change slightly, be specially:

Animal grouping: 4 blank groups, 3 positive controls, 6 groups of 18 average marks are as experimental group, and 3 every group, every group is designated as BC, PC, SP1, SP2, SP3, SP4, SP5, SP6 respectively;

Animal rearing: these SHR mouse are placed on 24 ± 1 ℃ of air constant temperature, and the room of humidity 50 ± 10% allows it accept illumination in 12 hours (morning 7:00～evening 7:00), and 12 hours dark are replaced in turn.Feed is conventional less salt or salt-free mouse grain, the tap water of water for filtering.Feed after 4 weeks, these SHR just can prepare administration.

2.2 animals administer

Synthetic peptide P1, P2, P3, P4, P5, P6 are mixed with the aqueous solution of 1mg/ml respectively; Amount by 10ml/kg is carried out gastric infusion to experimental group SP1, SP2, SP3, SP4, SP5, SP6 respectively; Blank group BC gives the pure water with dosage.

2.3 blood pressure determination

With adorning fixedly rat of mouse tube, expose tail; Preheating 25min in container, 36 ℃ of constant temperature are measured blood pressure with the non-invasive blood pressure tester from the rat caudal artery.Before the record administration, 3h after the administration, 15h, the pressure value of 24h (SBP).

2.4 statistical procedures

The SBP significant difference uses paired Student ' s t-test to analyze before and after the administration.

3, interpretation of result

The hypertensive rat blood pressure changing conditions

The SHRs gastric infusion is measured the blood pressure result and is shown that 6 synthetic peptide P1, P2, P3, P4, P5, P6 have stronger hypotensive activity in experimental animals, see Table 11.In administration after 3 hours, SP1, SP2, SP3, SP4, six groups of SP5, SP6 blood pressure lowering effect be (p＜0.05) obviously: the SBP (systolic pressure) of SP1, SP2, SP3, SP4, SP5, SP6 group descend respectively 22.5mmHg, 18.5mmHg, 15.1mmHg, 18.5mmHg, 5.85mmHg and 4.6mmHg.This shows to have after 3 hours at oral administration at administration polypeptide P1, P2, P3, P4, P5, P6 all significant hypotensive activity.After the administration 15 hours, the SBP (systolic pressure) of SP1, SP2, SP4 group descends still obviously (p＜0.05), but gos up to some extent.The SBP of SP3, SP5, SP6 group gos up more, still is in negative value, and this shows medicinal can the continuing for some time in vivo of polypeptide P1, P2, P4, and P3, P5, P6 effect metabolism are very fast.After 24 hours, the SBP of SP1, SP3, SP4, SP5, SP6 all recovered initial level, and the SP2 group was recovered initial level later at 36 hours.The disposable metabolism time that P1, P2, P3, P4, P5, P6 are described is 24～36 hours.Fig. 4,5,6,7 is seen in six groups of SBP variations.

4, conclusion and discussion

Respectively organize the SBP value (mmHg) of SHR before and after table 11 administration

Sample number	SBP before the administration	Behind the administration 3h	Behind the administration 15h	Behind the administration 24h	Behind the administration 36h
						BC	165.75±0.35	168.75±3.18	164.50±2.12	164.75±1.77	165.30±2.24
P1	154.75±6.72	131.75±0.35	139.50±1.84	153.65±5.16	155.82±2.12
						P2	163.55±5.73	144.70±2.97	146.15±2.62	155.5±4.24	162.03±4.33
P3	158.4±8.4	143.90±4.67	153.00±3.33	158.5±2.36	155.69±0.85
						P4	156.6±6.4	138.1±4.41	147.85±2.11	155.7±2.47	157.6±4.1
P5	162.85±5.37	157.0±4.17	161.25±5.31	162.4±2.3	164.05±2.11
						P6	159.2±5.7	154.6±2.11	157.3±2.17	158.8±3.25	161.2±3.11

Pharmacological evaluation shows that polypeptide P1, P2, P3, P4, the spontaneous hypertensive rat of P5, P6 (SHR) have significant hypotensive activity.Can bring into play drug effect in 1～36 hour after administration, hypotensive activity in 3 hours is the strongest after the administration.Wherein, the P1 blood pressure lowering effect is the most obvious, and the P2 blood pressure lowering effect time length is the longest, and P3 metabolism in vivo gets the fastest.

The present invention shows that by the external digestion experiment polypeptide peptide of the present invention acts on enhancing respectively after by stomach en-, trypsinase, stomach en-and trypsin digestion, infers that in conjunction with pharmacological evaluation six kinds of peptide hypotensive activities in vivo are relevant with digestive ferment in the body that participates in reaction.Normal conditions, polypeptide is oral to be entered after the human body, is tentatively digested by stomach earlier, contains a large amount of stomach en-s in the stomach, and the time of Ting Liuing is shorter under one's belt; Enter small intestine then, be absorbed then through the multiple digestive ferment digestion of pancreatic juice and small intestinal cell secretion, wherein the major protein enzyme is trypsinase.

P1, P4, P5, P6 are entering the first step of Digestive tract, just can directly absorbed by small intestine, and the P1 antihypertensive effect be the most obvious by the very strong little peptide of gastric pepsin digestion one-tenth effect.P2 need just can better play a role behind tryptic digestion in small intestine, and the reaction times is longer, so acting duration is also long.P3 can be digested simultaneously by GI enzyme, so metabolism in vivo is very fast.And these several peptide species might be precursor type inhibitor (Prodrug-type inhibitor), such polypeptide can be hydrolyzed to real inhibitor by ACE or gastrointestinal enzyme, still keeping antihypertensive activity after these polypeptide are oral by spontaneous hypertensive rat, its IC ₅₀Compare with parent peptide (digestive ferment handle before original polypeptide) and to diminish, effect is better.

Claims (3)

1. aminoacid sequence is the purposes of the described polypeptide of SEQ ID No.3 in the preparation Altace Ramipril, and described Altace Ramipril is oral pharmaceutical.

2. Altace Ramipril, it is characterized in that: containing aminoacid sequence is that the described polypeptide of SEQ ID No.3 is as effective constituent; Described Altace Ramipril is oral preparations.

3. Altace Ramipril according to claim 2, it is characterized in that: described oral preparations is capsule, tablet, dripping pill, oral liquid or granule.

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