CN101265292A - Polypeptides substances, preparing method and use thereof - Google Patents

Abstract

The invention discloses a polypeptide substance, preparation method and application thereof. The invention also discloses extraction process of two polypeptides, and determines molecular weight and amino acid sequence of the polypeptides. The polypeptides are synthesized by the chemical method, and purity analysis and molecular weight measurement results of the polypeptides indicate that molecular ion peak (m/z) completely satisfies theoretical molecular weight. Amino acid analysis result of the polypeptides indicates that amino acid content and the composition of the peptides are in accordance with theoretical values. Amino acid sequence measurement result of the polypeptides indicates primary structure thereof is in accordance with the designed sequence. At the same time, activity measurement result of the polypeptides indicates that the two peptides have activity, and remarkable effect on the treatment of myocardial ischemia, cerebral ischemia, and hepatic ischemia.

Description

Polypeptides matter, Its Preparation Method And Use

Technical field

The present invention relates to polypeptides matter, specifically two kinds of active polypeptide, Its Preparation Method And Use.

Background technology

" myocardial preservation " is the focus of the inside and outside section of heart research in recent years always.Data shows recently, in the myocardial cell of hypoxic-ischemic many variations can take place, and comprises intracellular calcium overload, free-radical generating, and membrane damage, ATP (adenosine triphosphate, ATP) level descends, and oxygen is exhausted or the like.

In order to intervene myocardial ischemia and protection cardiac muscle, studied many medicines over nearly 20 years, as beta-blocker, calcium antagonist, converting enzyme inhibitor, various oxygen free radical scavengers etc., but it is clinical as yet uncertainly to myocardial protection effect.The medicine of existing treatment myocardial ischemia does not also have a kind ofly can definitely reduce myocardial infarction, to resisting myocardial ischemia.

Existing all kinds of cardiovascular disease therapies medicine, remove converting enzyme inhibitor and have the generation of retardance somatomedin, arrestin matter is synthetic, alleviates outside the effect of myocardial hypertrophy (Hypertrophy), and other medicines all do not have the direct effect of regulating the cardiac muscle growth, breaking up, repair.In recent years, abroad begun to pay attention to using pharmaceutical methods to induce the protective capability of cardiac muscle self, promoted the research of myocardial cell's regeneration etc., the research of Cardiotrophin and Myotrophin as transducible gene.On the other hand, by the various pass through mechanism of extracellular signal triggering, the propagation or the reconstruct of regulation and control cardiac muscle, vascular cell.But all these researchs all are in animal experiment or preclinical study stage.

Above-mentioned research clearly proves: under the imperfect as yet situation of extracorporeal circulation methods of myocardial protection; design a kind of harmless to body; again can be before art, in the art and the medicine of postoperative protection cardiac muscle; to exploring the control of myocardial ischemia and reperfusion injury; provide new thinking and approach, with significant.

ZL94102798 discloses a kind of CMGSP and preparation method thereof, choose the heart of healthy young mammals, adopt that mechanical system is smashed to pieces ,-20 ℃ of dark freezing-dissolve post-heating 60-100 ℃, the centrifugal 3000rpm in ℃ dark freezing-dissolve back again-20, hold back post-degerming-packing-freeze-drying-packing through negative pressure, obtain molecular weight less than 20000 daltonian polypeptide class active substances.

ZL94102799 discloses a kind of CMGSP (GMGSP) with the synthetic and protein synthesis of the DNA that stimulates former generation cardiac muscle cells, is that preparation is extracted from the heart of healthy young mammals, and is stable in the pH2-9 scope; The heating 95-100 ℃ 10 minutes, 60-70 ℃ of 30 minutes following biological activity do not change; At the multiple protein lytic enzyme, loss of bioactivity under 37 ℃ of 2 hours conditions; Under 22 ℃ of-30 ℃ of conditions of the aqueous solution, can form polymer but biological activity changes not obvious; Adding under the 3%-8% N.F,USP MANNITOL freeze-drying air-proof condition, room temperature storage 1.5 years was stored 2 years for 4 ℃, stored 3 years for-20 ℃, and biological activity does not change; The HPLC analysis revealed: described GMGSP is made up of four components, and relative peak of each component and retention time are respectively: 10.4% (2.88 minutes), 6.4% (3.93 minutes), 36.3% (5.09 minutes), 7.3% (7.41 minutes), the equal biologically active of each component; Analyze the two band molecular weight that show through SDS-PAGE and be respectively 8500Da, 10800Da, HPLC analyzes number-average molecular weight 9800Da, weight-average molecular weight 10500Da, 2 components all have biological activity.

In Chinese invention patent 03141352.8 myocardium peptide and uses thereof, this disclosure of the Invention myocardium peptide be never to comprise in people's the health mammal heart extracting, wherein the content of polypeptide is 75%-90%, total free aminoacids is 6%-15%, rna content is 1%-2%, thymus nucleic acid content is 3%-7%, and weight-average molecular weight is 1000-1000 dalton.This invention also discloses the purposes of described myocardium peptide aspect preparation treatment cardiovascular disease medicine and myocardial ischemia and reperfusion injury medicine.And a kind of preparation method of myocardium peptide is disclosed in Chinese invention patent 03137133.7, its concrete grammar is: the health mammal ventricular muscles that will not comprise the people is cleaned, chopping, add sterile purified water homogenate, homogenate is freezing repeatedly, thaw 3-4 time, be heated to 65-95 ℃ of filter cleaner, obtain coarse filtration liquid with the filtration of sheet frame filter, using hollow fiber column ultrafilter, obtain smart filtrate, use the ultra-filtration membrane ultrafiltration, hold back the myocardium peptide solution of weight-average molecular weight, concentrate with the reverse osmosis concentration post less than 10000Da, degerming after filtration at last, lyophilize gets finished product.

But above-mentioned result of study shows, myocardium peptide is never to comprise in people's the health mammal heart extracting, and wherein contains various active compositions such as polypeptide, total free aminoacids, thymus nucleic acid,

Polypeptide be a class by each seed amino acid, the protein-based compound of forming by different series arrangement of a kind of catenate.This compounds often has the very strong physiologically active and the specificity of function.Therefore, these polypeptides matters having many uses in some disease medicament of preparation treatment is general.

Yet up to now, for the sequence of polypeptide contained in the myocardium peptide and all relevant patent of purposes and bibliographical information.

Summary of the invention

The object of the invention is to provide two peptide species, and wherein the sequence of a peptide species is:

Trp-Ser-Asn-Val-Leu-Arg-Gly-Met-Gly-Gly-Ala-Phe, molecular weight were 1294.75 (being called for short myocardium peptide X); On the basis of aforementioned polypeptides,, form a kind of new polypeptide (being called for short myocardium peptide C) by connecting L-Ala, glycine, Methionin successively at an end that is connected with tryptophane, it is amino acid whose put in order into:

Lys-Gly-Ala-Trp-Ser-Asn-Val-Leu-Arg-Gly-Met-Gly-Gly-Ala-Phe, molecular weight are 1550.91.Extract in the described polypeptide cardiac muscle peptide solution, aforementioned polypeptides has activity after measured, and comprises that as preparation treated tissue organ ischemic myocardial ischemia, cerebral ischemia, hepatic ischemia, stomach ischemic, intestinal ischemia, ischemia of lung, renal ischaemia prescription face purposes are remarkable.

To achieve these goals, the technical solution used in the present invention is:

One peptide species, described polypeptide amino acid whose put in order into:

Trp-Ser-Asn-Val-Leu-Arg-Gly-Met-Gly-Gly-Ala-Phe。

An end that is connected with tryptophane in aforementioned polypeptides connects L-Ala, glycine, Methionin successively, forms a kind of new polypeptide, it is amino acid whose put in order into:

Lys-Gly-Ala-Trp-Ser-Asn-Val-Leu-Arg-Gly-Met-Gly-Gly-Ala-Phe。

Another object of the present invention is to provide the extracting method of a kind of myocardium peptide C.

To achieve these goals, the technical solution used in the present invention is:

A kind of method of extracting myocardium peptide C from myocardium peptide solution, described method comprises the steps:

(1) add ammoniumsulphate soln at myocardium peptide solution, place, centrifugal, separation of supernatant and precipitation;

(2) get supernatant liquor, cross solid phase extraction column, use the organic solvent wash-out, collect fraction A;

(3) fraction A of collecting is concentrated, separate with reversed-phase preparative chromatography then, collect activated fraction B;

(4) activated fraction B is concentrated, further separate with the analysis mode reverse-phase chromatography then, collect activated cut C;

(5) activated cut C is carried out mass spectrometric detection, by MASS SPECTRAL DATA ANALYSIS, identify the aminoacid sequence of activated peptide section among the cut C, described cut C is described polypeptide.

The mass percent concentration of ammoniumsulphate soln of the present invention is 60-80%, and preferred described concentration is 70%.

Organic solvent described in the step of the present invention (2) is the mixing solutions of 60% acetonitrile and 0.1% trifluoroacetic acid.

In the step of the present invention (3) fraction A is concentrated into the 1/8-3/4 of original volume; Utilize the isolating condition of reversed-phase preparative chromatography to be: moving phase: A is 100%H mutually ₂O+0.1%TFA, B are 60%ACN+0.1%TFA mutually; UV-detector, wavelength X=280nm, R=3.0; Gradient elution: 0-5min0%B, 5-35min0%-100%B, 35-40min100%B, 40-45min100%-0%B.

Step of the present invention is concentrated into 1/8-1/2 with fraction B in (4); Describedly further be separated at chromatographic condition as follows with the analysis mode reverse-phase chromatography: moving phase: A is 100%H mutually ₂O+0.1%TFA, B are 95%ACN+0.1%TFA mutually; UV-detector, wavelength X=280nm, R=3.0; Gradient elution: 0-5min0%B, 5-50min0%-100%B, 50-60min100%B carries out under the condition of 60-65min 100%-0%B.

A further object of the present invention is to provide the chemical synthesis process of a kind of myocardium peptide C.

To achieve these goals, the technical solution used in the present invention is:

A kind of chemical synthesis process for preparing myocardium peptide C, described method comprises:

(1) respectively tryptophane, Serine, l-asparagine, Xie Ansuan, leucine, arginine, glycine, methionine(Met), L-Ala, phenylalanine, lysine amino are protected with blocking group;

(2) earlier with the solid phase carrier swelling, again the amino acid after first protection in the sequence of amino acid of polypeptide is fixed on this solid phase carrier, then products therefrom is washed, obtain being connected with the amino acid whose solid phase carrier after first protection;

(3) protect amino acid to join with second and be combined with in first amino acid whose solid phase carrier, react the formation peptide bond in the presence of condensing agent, the blocking group with amino acid whose amino removes and washs resulting solid phase carrier then;

(4) according to the amino acid ordering of polypeptide, add corresponding protection amino acid successively, the step that the blocking group that repeats above-mentioned amino removes, washs constantly prolongs peptide chain, till to the last an amino acid is attached on the peptide chain;

(5) get the solid phase carrier that is combined with polypeptide that parting liquid soaks above-mentioned steps (4) gained, precipitate through getting gained filtrate after filtering then, obtain the polypeptide crude product;

(6) the polypeptide crude product with gained carries out separation and purification, obtains target product.

The described amino acid whose amino of step of the present invention (1) can be protected by following group:

(1) carbobenzoxy (Carbobenzoxyl)

(2) uncle-butoxy carbonyl (t-Butyloxycarbonyl)

(3) tosyl group (Tosyl)

(4) 9-fluorenylidene methoxycarbonyl (Fmoc:9-Fluorenylmethoxycarbonyl).

At each polypeptide of occurring in nature or protein all is to be polymerized by the form of free amino acid by the polypeptide key.In proteinic translation process, the extension of polypeptide key is always carried out to carboxyl terminal (C-end) by aminoterminal (N-end).When chemosynthesis begins; elder generation is fixed to first amino acid of carboxyl terminal on the activatory resin often; pass through the protection of going of amino group then; second amino acid with carboxyl terminal joins in the reactor again; two amino acid can form peptide bond through reacting to each other; amino acid collating sequence according to polypeptide adds the 3rd, the 4th amino acid or the like successively then, till to the last an amino acid is attached on the peptide chain.

The present invention also has a purpose to be to provide myocardium peptide C to prevent and/or treat the purposes of histoorgan ischemic prescription face in preparation, preferred described polypeptide prevents and/or treats the purposes of myocardial ischemia, cerebral ischemia, hepatic ischemia, stomach ischemic, intestinal ischemia, ischemia of lung or renal ischaemia prescription face in preparation, and more preferably described polypeptide prevents and/or treats the purposes of myocardial ischemia prescription face in preparation.

The invention has the beneficial effects as follows: the invention discloses the extracting method of two peptide species, and disclose two peptide species and sequence contained in the myocardium peptide solution, finished chemosynthesis, purifying, determination of activity and the evaluation of this two peptide species.Determination of activity shows that this two peptide species has activity, shows through experimentation on animals and clinical study, and this two peptide species is remarkable as preparation treatment myocardial ischemia, cerebral ischemia and the effect of hepatic ischemia prescription face.Simultaneously these two kinds have structure clear and definite, active high, easily produce, cost is low and be easier to research sets forth characteristics such as its mechanism of action.

Description of drawings

Fig. 1: molecular weight is 1294.75 (myocardium peptide X) mass spectrum (4700MS/MS Procursor 1294.75Spec#1MC[BP=1147.4,4886])

Fig. 2: molecular weight is 1550.91 (myocardium peptide C) mass spectrum (4700MS/MS Procursor 1550.91Spec#1MC[BP=1403.5,3630])

Fig. 3: molecular weight is the secondary spectrum denovo sequences match KGAWSNVLRGMGGAF of 1550.91 (myocardium peptide C)

Fig. 4: molecular weight is the secondary spectrum denovo sequences match WSNVLRGMGGAF of 1294.75 (myocardium peptide X)

Fig. 5: the color atlas of cut C

Fig. 6: the extracting method particular flow sheet of polypeptide (myocardium peptide C)

Fig. 7: myocardium peptide C preventive administration is to the influence of ischemia-reperfusion rat heart muscle infarct size, Fig. 7-a, sham operated rats; Fig. 7-b is a model group; The positive medicine group of Fig. 7-c; Fig. 7-d is the CMPC group

Fig. 8: the administration of myocardium peptide C therapeutic is to the influence of ischemia-reperfusion rat heart muscle infarct size, Fig. 8-a, sham operated rats; Fig. 8-b is a model group; The positive medicine group of Fig. 8-c; Fig. 8-d is the CMPC group

Embodiment

Embodiment 1

Present embodiment relates to the synthetic of polypeptide (myocardium peptide C).

The sequence of described polypeptide is Lys-Gly-Ala-Trp-Ser-Asn-Val-Leu-Arg-Gly-Met-Gly-Gly-Ala-Phe

1, synthetic: as at first amino acid whose amino contained in the polypeptide to be protected (consumption 20-100 gram/gram) with amido protecting group (9-fluorenylidene methoxycarbonyl); the wang resin is carried out swelling with chlorethyl ether; the consumption of used chlorethyl ether is 50-100 milliliter/gram; then first amino acid is joined in this resin, thereby it is fixed on the resin.Through after the washed with dichloromethane, amino blocking group can use hexahydropyridine (consumption 10-80%, preferably 20-40%) deprotection to get up.Meanwhile, with second amino acid whose carboxyl process HBTU[2-(1 hydrogen-benzotriazole base)-1,1,3,3-tetramethyl-alditol-hexafluoro closes phosphoric acid ester] in activation and the diisopropylethylamine and after, join and be combined with in first amino acid whose resin, can form peptide bond after two amino acid react.According to the amino acid ordering of peptide material, add corresponding amino acid successively then, repeat above-mentioned washing; amino goes protection; the steps such as activation of amino acid carboxyl connect reactive polypeptide to carry out 14 times, thereby polypeptide chain is constantly prolonged, till to the last an amino acid is attached on the peptide chain.Polypeptide after synthetic still with resin-bonded together, utilize parting liquid that peptide material is dissociated to get off from resin.Parting liquid can adopt phenol, thioanisole and trifluoroacetic acid one or more mixture wherein, the parting liquid of getting the 10-80 milliliter soaks the above-mentioned resin that is combined with polypeptides matter (II), at room temperature stirred 1.5 hours, just can obtain filtrate through filtration then is the peptide material mixed solution.The free peptide material can be separated through behind the ether sedimentation.The peptide material of separating is dissolved in the alkaline solution of water or 10-40% (alkali can be selected for use: bicarbonate of ammonia, volatile salt, sodium bicarbonate, yellow soda ash), uses conventional liquid phase chromatography then, complete peptide material can be carried out purifying.Whether the peptide material behind the purifying can record its concentration and can examine the aminoacid sequence of institute's synthetic polypeptides matter again by mass spectroscopy correct by ultraviolet spectrophotometry.

Embodiment 2

Present embodiment relates to the chemical synthesis process of myocardium peptide X.

The aminoacid sequence of described myocardium peptide C is: Trp-Ser-Asn-Val-Leu-Arg-Gly-Met-Gly-Gly-Ala-Phe.

Used method such as embodiment 1.

Embodiment 3

Present embodiment relates to the chemical synthesis process of myocardium peptide C.

The sequence of described polypeptide is Lys-Gly-Ala-Trp-Ser-Asn-Val-Leu-Arg-Gly-Met-Gly-Gly-Ala-Phe.

Described method may further comprise the steps:

(1) removes protecting group Fmoc: get Fmoc-Lys (Boc)-CLTR resin; with methylene dichloride washing by soaking 3 times; each 2min drains then, at room temperature adds the dichloromethane solution 10mL of 50% piperidines; drain behind the reaction 5min; still the solution with above-mentioned same amount reacts 30min again, drains, afterwards successively with methylene dichloride, ethanol and the washed with dichloromethane of 8mL～10mL again; drain the resin that takes a morsel (2mg～10mg) measure total amino amount with the quantitative free radical method of salicylic aldehyde.

(2) formation of peptide bond (DCC-HOBt condensation method): in the resin of the above-mentioned Fmoc of going protecting group, add the dichloromethane solution of Fmoc-Gly (t-Bu) and the dichloromethane solution of 1-hydroxy benzo triazole; and after adding dimethyl formamide solution hydrotropy 30min simultaneously; add N; the dichloromethane solution 2mL of N '-dicyclohexylcarbodiimide; reaction is spent the night under 20 ℃; drain, washing gets resin peptide Fmoc-Gly (t-Bu)-Lys (Boc)-R.Take a morsel resin drying to constant weight, measure the residual ammonia base unit weight with the quantitative free amino group method of salicylic aldehyde, and calculate the condensation rate thus.In reaction process, carry out degree with the ninhydrin monitoring reaction, when resin was the water white transparency shape, the expression condensation reaction was finished.

(3) the not acetylize of condensation amino: the resin peptide 30min of the gained pyridine solution treatment step 2 under room temperature that contains 50% aceticanhydride with 10mL), washing is drained, and handles once with same solution and amount again, makes the not glycylization of condensation.

(4) resin peptide Fmoc-Lys (the Boc)-Wang-R in the step (1) is changed to Fmoc-Gly (t-Bu)-Lys (Boc)-R, Fmoc-Gly (t-Bu) in the step (2) is changed to Fmoc-Ala (Trt), the operation of repeating step (1)～step (3), by that analogy, be about to back synthetic resin peptide and make next step synthetic amino group, carry out 14 times and connect reactive polypeptide, all form to the peptide bond between 15 amino acid;

(5) remove resin on Side chain protective group and the peptide chain: in the end in 12 peptide resins of Xing Chenging, add 30mL methylene dichloride, trifluoroacetic acid 3.0mL, in 25 ℃ of reaction 60min, filter, filtrate keeps stand-by.The dichloromethane solution 10mL that will contain 50% trifluoroacetic acid adds filtrate, reaction 20min, use above-mentioned solution washing 3 times afterwards, washing lotion and last time filtrate merger, through Rotary Evaporators steam to surplus liquid a little, add a large amount of anhydrous diethyl ethers, separate out the white powder thing, liquid phase is removed in centrifugation, with anhydrous diethyl ether powder is ground 5 times afterwards, the final vacuum drying, the calculating productive rate of weighing;

(6) product that takes a morsel carries out electrophoresis detection and RP-HPLC and analyzes, and chromatographic condition is: Alltech PlatinumC18 chromatographic column; Buffer A is 0.1% trifluoroacetic acid (TFA) aqueous solution; Buffer B is the aqueous solution of the second eyeball (more than be all volume fraction) of 0.1% TFA and 90%; Gradient is that the volume fraction of buffer B rises to 50% from 10% in the 40min; Flow rate of mobile phase is that 1.0ml/min detection wavelength is 214nm.

Embodiment 4

Present embodiment relates to the synthetic of polypeptide (myocardium peptide X)

The sequence of polypeptide is Trp-Ser-Asn-Val-Leu-Arg-Gly-Met-Gly-Gly-Ala-Phe

Used method such as embodiment 3.

Embodiment 5

Present embodiment relates to the method for extracting polypeptide (myocardium peptide C) from myocardium peptide solution.

(1) plant and instrument

According to sharp extra-high voltage liquid chromatography P230 system; The two-dimensional liquid chromatography separation system of PF-2 (U.S. Dai An company) analysis mode chromatographic column; Waters, C18,4.6 * 250mm, SPE solid phase extraction column (waters company); C18 partly prepares reverse-phase chromatographic column (U.S. power ﹠ light company); Vacuum frost drying machine (U.S. power ﹠ light company); Whizzer etc.

(2) experiment condition and method

1. adding 70% ammoniumsulphate soln at myocardium peptide solution, is to place under 4 ℃ the condition to spend the night in temperature, centrifugal, separation of supernatant and precipitation;

2. get supernatant solution, cross the SPE solid phase extraction column,, collect fraction A with the mixing solutions wash-out of 60% acetonitrile and 0.1% trifluoroacetic acid;

3. the fraction A of collecting is concentrated, separate with reversed-phase preparative chromatography then, collect activated fraction B;

Chromatographic condition is as follows:

Moving phase: A is 100%H mutually ₂O+0.1%TFA, B are 60%ACN+0.1%TFA mutually

UV-detector, wavelength X=280nm, R=3.0

Gradient elution: 0-5min0%B, 5-35min0%-100%B, 35-40min100%B, 40-45min100%-0%B;

4. activated fraction B is concentrated, further separate with the analysis mode reverse-phase chromatography then, collect activated cut C;

Chromatographic condition is as follows:

Moving phase: A is 100%H mutually ₂O+0.1%TFA, B are 95%ACN+0.1%TFA mutually

UV-detector: wavelength X=280nm, R=3.0

Gradient elution: 0-5min0%B, 5-50min0%-100%B, 50-60min100%B, 60-65min100%-0%B

5. activated cut C is carried out mass spectrometric detection,,, identify the aminoacid sequence that activated peptide section among the cut C is arranged as Fig. 2-shown in Figure 3 by MASS SPECTRAL DATA ANALYSIS.

Embodiment 6

The structure that present embodiment relates in the process of extracting polypeptide (myocardium peptide C) from myocardium peptide solution is identified and quality examination.

(1) mass spectrometric detection

Utilize MALDI-TOF-TOF mass spectrograph (American AB company-4800) that products therefrom is carried out mass spectrometric detection, wherein MALDI-TOF one-level mass spectrum adopts positive ion reflection detecting pattern; The two-stage tandem mass spectrum adopts the positive ion reflective-mode, and detected result as shown in Figure 2.

(2) the active detection

" myocardial cell cultivates active determination test " method that determination of activity adopts Dalian Zhen Ao Pharma Inc. to provide is carried out.This method is on vitro culture myocardial cell Zorubicin poisoning experiment basis, and with 2,4-phenylbenzene bromination tetrazolium (MTT) makes the effect of damaged myocardium cell mitochondrial dehydrogenase activity enhanced as reaction indicator to observe myocardium peptide, to judge myocardium protecting action.

The result of determination of activity is as shown in the table

Sample number into spectrum	Former state	The peptide section	The peptide section	The peptide section
					Activity value	3.2437	2.3570	2.7328	2.564

Embodiment 7

Present embodiment relates to the purposes of myocardium peptide C (CMPC) at preparation and/or treatment myocardial ischemia prescription face.

One, experiment material

1. experimental animal

SD rat suckling mouse, the 2-4 age in days is provided by Shenyang Pharmaceutical University's animal center.

The SD rat, body weight 200～300g, male and female half and half are provided by Academy of Military Medicine, PLA experimental animal center.Animal conformity certification numbering: SCXK (army) 2002-001.

2. for reagent thing and reagent

Cardiac muscle peptide C (CMPC) is provided lot number: C1586102 by Zhenao Pharmaceutical Co., Ltd., Dalian City;

Verapamil injection liquid (Ver): Shanghai Hefeng Pharmaceutical Co., Ltd., specification: 2ml:5mg, lot number: 070701;

Injection Dx (Zorubicin): Shantou, Guangdong special zone Mingzhi medicine company limited, specification: 10mg/ bottle, lot number: 070202;

DMEM substratum (high sugar): Gibco, lot number: 1290007;

DMEM substratum (low sugar): Gibco, lot number: 1348529;

II Collagen Type VI enzyme: Gibco

Foetal calf serum: Tianjin Hao ocean biological products science and technology responsibility company limited, lot number: 04700701-3100

Tetramethyl-azo azoles indigo plant (MTT): Amresco

NBT:Amresco, specification: 100mg

Urethane: Tianjin benchmark chemical reagent company limited, lot number: 20060218;

Superoxide-dismutase (SOD), mda (MDA), creatine kinase (CK) and serum lactic dehydrogenase (LDH) test kit all build up bio-engineering research institute, lot number: 20080122 available from Nanjing;

Glacial acetic acid: Tianjin chemical industry all generations company limited, specification: 500ml, lot number: 20070712;

Dehydrated alcohol: Tianjin chemical industry all generations company limited, specification: 500ml, lot number: 20070410

Two, experimental technique

1. in vitro tests

1.1 the myocardial cell is former be commissioned to train foster

Get rat suckling mouse ventricle, D-Hank ' s shreds after cleaning, and with the enzymic digestion of II Collagen Type VI, obtains myocardial cell and inoblast, cultivates with the high sugared DMEM (DMEM-H-15) that contains 15% foetal calf serum.Adherent and chemical process removal inoblast is inoculated into the myocardial cell in 96 orifice plates by differential.

Myocyte's Zorubicin damaging action that nourishes heart 1.2.CMPC antigen is commissioned to train

Cell culture processes is with 1.1.

The model preparation: the blank group is established in experiment: the myocardial cell cultivates with DMEM-H-15; Model group: add the 1mg/L Zorubicin, behind the cultivation 1h, wash plate 2 times, add DMEM-H-15 and cultivate with DMEM-H-15; Positive controls (verapamil 2,0.4,0.08mg/L) and different concns are subjected to reagent thing group ( CMPC 50,10,2,0.4,0.08,0.016,0.0032,0.00064mg/L): the myocardial cell who Zorubicin is damaged 1h adds each concentration verapamil injection liquid (Ver) and CMPC (the above-mentioned culture system cubic capacity of respectively organizing is 100 μ l/ holes, and drug level all refers to final concentration) respectively after washing plate 2 times with DMEM-H-15.Measure mitochondrial succinate dehydrogenase vigor in the myocardial cell with mtt assay after continuing to cultivate 24h, and calculate protection ratio, ask and calculate EC ₅₀Value.

Protection ratio=(A _x-A _Model)/(A _Control-A _Model) * 100%

A _x, A _Model, A _ControlThe absorbance of representing each administration group, model group and blank group respectively.

In the Zorubicin damage model, CMPC can significantly improve myocardial cell's survival rate, and its protection effect was better when drug level was 0.016-2mg/L, had compared significant difference with model group.The EC of CMPC in the culture plate 1,2,3 in the Zorubicin damage model ₅₀Be respectively 0.15,0.13,0.27mg/L.The results are shown in Table for 1 (CMPC represents with " % " myocardial cell's protection ratio).

The myocardium peptide C of table 1 is to the influence of myocardial cell's survival rate of Zorubicin damage

^*P＜0.01, ^*P＜0.05 and model group contrast ^##P＜0.01, ^#P＜0.05 and the contrast of blank group

The oxygen supply damaging action of myocyte's anoxic-again that nourishes heart 1.3.CMPC antigen is commissioned to train

Myocardial cell's cultural method is with 1.1.

Model preparation: get the myocardial cell who cultivates 48-72h and prepare model, each porocyte in the culture plate is divided into control group, model group, verapamil group (2,0.4,0.08mg/L final concentration) and CMPC group (final concentration be 50,10,2,0.4,0.08,0.016,0.0032mg/L), and each group is 3 multiple holes.Except that control group, all the other cells prepare model by the following method: after using myocardial cell's individual layer in the culture plate instead low sugar culture-medium (including the verapamil or the CMPC of respective concentration), culture plate is placed vacuum drier, charge into nitrogen, airtight then cultivation; Anoxic 15h causes myocardial cell injury; Use high sugared DMEM nutrient solution (95% air+5%CO after the anoxic instead ₂) cultivate 1h, cause and give oxygen injury again.With the absorbance at mtt assay mensuration 540nm place, the same calculating protection ratio is asked and is calculated EC ₅₀Value.

In anoxic reoxygenation injury model, CMPC can significantly improve myocardial cell's survival rate, and its protection effect was better when drug level was 0.08-2mg/L, had compared significant difference with model group, the EC of CMPC in the culture plate 1,2,3 ₅₀Be respectively 0.66,0.14,0.82mg/L.The results are shown in Table for 2 (CMPC represents with " % " myocardial cell's protection ratio).

The myocardium peptide C of table 2 is to the influence of anoxic-reoxygenation induced injured myocardium cell survival rate

^*P＜0.01, ^*P＜0.05 and model group contrast ^##P＜0.01, ^#P＜0.05 and the contrast of blank group

2. experiment in the body

2.1.CMPC preventive administration is to the provide protection of rat heart muscle ischemia reperfusion injury

Get 70 of 250-300g SD rats, wherein 60 are divided into 6 groups at random by the body weight equilibrium, 10 every group, remain 10 and respectively organize dead animal in order to replenishing.The normal control group: the row sham-operation, promptly coronary artery left anterior descending branch (LAD) locates to wear a silk thread, but refuses ligation; Model group: 30min is through intravenous injection physiological saline 5ml/kg before the ligation, and the ischemia-reperfusion operation sees the model preparation for details.The positive drug group: 30min is through intravenous injection verapamil 1.0mg/kg before the ligation, and ischemia-reperfusion is operated same model group; The high, medium and low dosage group of CMPC: 30min is through intravenous injection respective concentration CMPC (1.8,0.6,0.2mg/kg) before the ligation, and ischemia-reperfusion is operated same model group.

Model prepares: after pressing 20ml/kg intraperitoneal injection of anesthesia animal with 6% urethane solution, the animal dorsal position is fixed on the operating table; Connect electrocardiograph, survey limbs II lead electrocardiogram; Trachea cannula meets the animal breathing apparatus, presses the frequency of 6ml/200g Tidal volume, 55 times/min and gives and the end-tidal continuous positive pressure ventilation, and respiratory quotient is 2: 1.In left border of sternum the four/five intercostal space row throacotomy, after the separation rib is removed pericardium, be sign with the left auricle of heart lower edge, in the left anterior descending coronary artery ligation, between ligature and blood vessel, put the polyethylene tube that a bit of diameter is 1mm with 6/0 silk thread; Silk ligature makes polyethylene tube compressing left anterior descending coronary artery cause myocardial ischemia.Lose color and electrocardiogram(ECG S-T section is raised expression left anterior descending coronary artery ligation success with local myocardial.The careful polyethylene tube that takes out causes multiple filling behind the 30min.Confirm multiple the filling successfully more than 1/2 by ischemic region reactive hyperemia and the S-T section decline of raising.Irritate 30min again, abdominal aortic blood, preparation serum is used to measure biochemical indicator (press the test kit specification sheets and measure CK, LDH, MDA, SOD content).

Get to cut open behind the blood and get rat heart, clean hematocele in the chambers of the heart with physiological saline, claim heavyly whole-heartedly, remove fat, myocardium crosscut 4-5 sheet is placed test tube, 37 ℃ of descending NBT dyeing (healthy tissues dyeing, ischemic tissue does not dye) are taken pictures.The cutting-out ischemic myocardium is weighed, with the Weight of Ischemic Myocardium and the percentage calculation myocardial infarction area (MIS) of weight in wet base whole-heartedly.

Model group rat blood serum CK, LDH significantly raise (P＜0.01); The positive drug group significantly reduces serum CK, LDH activity (P＜0.05); The dosage group is compared with model group among CMPC high dose group, the CMPC, can reduce rat blood serum LDH activity (P＜0.05) significantly; The dosage group can significantly reduce CK activity (P＜0.05) among CMPC high dose group, the CMPC; The CMPC low dose group can reduce the activity of LDH, CK, but there was no significant difference (P＞0.05).The results are shown in Table 3.

The myocardium peptide C of table 3 preventive administration is to the influence of ischemia-reperfusion rat blood serum CK, LDH (x ± SD)

^*P＜0.05, ^*P＜0.01 and model group contrast; ^##P＜0.01 and sham operated rats contrast

Active significantly reduce (P＜0.01) of model group rat blood serum SOD, the MDA value is rising (P＜0.01) significantly; The positive drug Ver activity of SOD in serum (P＜0.01) that can significantly raise reduces MDA activity (P＜0.05); CMPC-H, M, L dosage group and model group relatively all can significantly improve coronary ligation rat blood serum SOD vigor (P＜0.01, P＜0.05), reduce MDA content (P＜0.01, P＜0.05).The results are shown in Table 4.

The myocardium peptide C of table 4 prevention administration is to the influence of ischemia-reperfusion rat blood serum SOD, MDA (x ± SD)

^*P＜0.05, ^*P＜0.01 and model group contrast; ^##P＜0.01 and sham operated rats contrast

Model group rat heart muscle infarct size significantly raises (P＜0.01), and positive drug group, each preventive administration group of CMPC and model group relatively all can reduce rat heart muscle infarct size (P＜0.01) by highly significant, the results are shown in Table 5, Fig. 7.

The myocardium peptide C of table 5 preventive administration is to the influence of ischemia-reperfusion rat heart muscle infarct size (x ± SD)

^*P＜0.05, ^*P＜0.01 and model group contrast; ^##P＜0.01 and sham operated rats contrast

2.2CMPC the therapeutic administration is to the provide protection of rat heart muscle ischemia reperfusion injury

5min irritates 30min again in the tail vein injection administration behind the ligation LAD behind the ligation LAD30min, cuts open after the aorta abdominalis blood sampling to core dirtyly, and the same method is measured biochemical indicator and myocardial infarction area.

Model group rat blood serum CK, LDH significantly raise (P＜0.01); Positive drug Ver significantly reduces serum CK, LDH activity (P＜0.05, P＜0.01); CMPC-H, M dosage group are compared with model group, can reduce rat blood serum CK, LDH activity (P＜0.01, P＜0.05) significantly; CMPC-L can reduce the activity of LDH, CK, but there was no significant difference (P＞0.05).The results are shown in Table 6.

The administration of the myocardium peptide C of table 6 therapeutic is to the influence of ischemia-reperfusion rat blood serum CK, LDH (x ± SD)

^*P＜0.05, ^*Compare with model group P＜0.01; ^##Compare with sham operated rats P＜0.01

Active significantly reduce (P＜0.05) of model group rat blood serum SOD, the MDA value is rising (P＜0.01) significantly; The positive drug Ver activity of SOD in serum (P＜0.05) that can significantly raise reduces MDA content (P＜0.05); CMPC-H, M and model group relatively all can significantly improve rat blood serum SOD vigor (P＜0.05, P＜0.01), and CMPC-L has the active trend of increased SOD, but does not relatively have significant difference (P＞0.05) with model group; CMPC-H, M, each dosage group of L all can reduce MDA content (P＜0.05), the results are shown in Table 7.

The administration of the myocardium peptide C of table 7 therapeutic is to the influence of ischemia-reperfusion rat blood serum SOD, MDA (x ± SD)

^*P＜0.05, ^*P＜0.01 compares with model group; ^##P＜0.01 compares with sham operated rats

Model group rat heart muscle infarct size significantly raises (P＜0.01), and positive drug Ver, CMPC therapeutic administration group and model group relatively all can reduce rat heart muscle infarct size (P＜0.05) by highly significant.The results are shown in Table 8, Fig. 8.

The administration of the myocardium peptide C of table 8 therapeutic is to the influence of ischemia-reperfusion rat heart muscle infarct size (x ± SD)

^*P＜0.05, ^*P＜0.01 compares with model group; ^##P＜0.01 compares with sham operated rats

Three, conclusion

Support in the damage of neonatal rat myocardial cell Zorubicin and the anoxic-reoxygenation injury model external former being commissioned to train, can the raise activity of myocardial cell's mitochondrial dehydrogenase of damage of myocardium peptide C has the certain protection effect to the myocardial cell of damage; Preventative and the therapeutic administration of body heart carnosine C all can significantly reduce ischemia-reperfusion rat blood serum CK, LDH, MDA content; remarkable increased SOD content; and certain dose-dependently is arranged; and can reduce the rat heart muscle infarct size, show that myocardium peptide C therapeutic and preventive administration all have provide protection to the rat heart muscle of ischemical reperfusion injury.

Sequence table

＜110〉Zhenao Pharmaceutical Co., Ltd., Dalian City

＜120〉contained active polypeptide and its production and use in the myocardium peptide

<130>5624-096

<160>2

<170>PatentIn version 3.1

<210>1

<211>12

<212>PRT

＜213〉artificial sequence

<400>1

Trp Ser Asn Val Leu Arg Gly Met Gly Gly Ala Phe

1 5 10

<210>2

<211>15

<212>PRT

＜213〉artificial sequence

<400>2

Lys Gly Ala Trp Ser Asn Val Leu Arg Gly Met Gly Gly Ala Phe

1 5 10 15

Claims (10)

1, a peptide species, it is characterized in that described polypeptide amino acid whose put in order into:

Trp-Ser-Asn-Val-Leu-Arg-Gly-Met-Gly-Gly-Ala-Phe。

2, polypeptide according to claim 1 is characterized in that an end that is connected to tryptophane at described polypeptide chain connects L-Ala, glycine, Methionin successively, it is amino acid whose put in order into:

Lys-Gly-Ala-Trp-Ser-Asn-Val-Leu-Arg-Gly-Met-Gly-Gly-Ala-Phe。

3, a kind of method of extracting the described polypeptide of claim 2 from myocardium peptide solution is characterized in that described method comprises the steps:

(1) add ammoniumsulphate soln at myocardium peptide solution, place, centrifugal, separation of supernatant and precipitation;

(2) get supernatant liquor, cross solid phase extraction column, use the organic solvent wash-out, collect fraction A;

(3) fraction A of collecting is concentrated, separate with reversed-phase preparative chromatography then, collect activated fraction B;

(4) activated fraction B is concentrated, further separate with the analysis mode reverse-phase chromatography then, collect activated cut C;

(5) activated cut C is carried out mass spectrometric detection, by MASS SPECTRAL DATA ANALYSIS, identify the aminoacid sequence of activated peptide section among the cut C, described cut C is described polypeptide.

4, method according to claim 3, the mass percent concentration that it is characterized in that described ammoniumsulphate soln is 60-80%, preferred described concentration is 70%.

5, method according to claim 3 is characterized in that the organic solvent described in the step (2) is the mixing solutions of 60% acetonitrile and 0.1% trifluoroacetic acid.

6, method according to claim 3 is characterized in that in the step (3) fraction A being concentrated into the 1/8-3/4 of original volume; Utilize the isolating condition of reversed-phase preparative chromatography to be: moving phase: A is 100%H mutually ₂O+0.1%TFA, B are 60%ACN+0.1%TFA mutually; UV-detector, wavelength X=280nm, R=3.0; Gradient elution: 0-5min0%B, 5-35min0%-100%B, 35-40min100%B, 40-45min100%-0%B.

7, method according to claim 3 is characterized in that in the step (4) fraction B being concentrated into 1/8-1/2; Describedly further be separated at chromatographic condition as follows with the analysis mode reverse-phase chromatography: moving phase: A is 100%H mutually ₂O+0.1%TFA, B are 95%ACN+0.1%TFA mutually; UV-detector, wavelength X=280nm, R=3.0; Gradient elution: 0-5min0%B, 5-50min0%-100%B, 50-60min100%B carries out under the condition of 60-65min 100%-0%B.

8, a kind of chemical synthesis process for preparing the described polypeptide of claim 2 is characterized in that described method comprises:

(1) respectively tryptophane, Serine, l-asparagine, Xie Ansuan, leucine, arginine, glycine, methionine(Met), L-Ala, phenylalanine, lysine amino are protected with blocking group;

(2) earlier with the solid phase carrier swelling, again the amino acid after first protection in the sequence of amino acid of polypeptide is fixed on this solid phase carrier, then products therefrom is washed, obtain being connected with the amino acid whose solid phase carrier after first protection;

(3) protect amino acid to join with second and be combined with in first amino acid whose solid phase carrier, react the formation peptide bond in the presence of condensing agent, the blocking group with amino acid whose amino removes and washs resulting solid phase carrier then;

(4) according to the amino acid ordering of polypeptide, add corresponding protection amino acid successively, the step that the blocking group that repeats above-mentioned amino removes, washs constantly prolongs peptide chain, till to the last an amino acid is attached on the peptide chain;

(5) get the solid phase carrier that is combined with polypeptide that parting liquid soaks above-mentioned steps (4) gained, precipitate through getting gained filtrate after filtering then, obtain the polypeptide crude product;

(6) the polypeptide crude product with gained carries out separation and purification, obtains target product.

9, chemical synthesis process according to claim 8 is characterized in that the described amino acid whose amino of step (1) can be protected by following group

(1) carbobenzoxy (Carbobenzoxyl)

(2) uncle-butoxy carbonyl (t-Butyloxycarbonyl)

(3) tosyl group (Tosyl)

(4) 9-fluorenylidene methoxycarbonyl (Fmoc:9-Fluorenylmethoxycarbonyl).

10, the described polypeptide of claim 2 prevents and/or treats the purposes of histoorgan ischemic prescription face in preparation, preferred described polypeptide prevents and/or treats the purposes of myocardial ischemia, cerebral ischemia, hepatic ischemia, stomach ischemic, intestinal ischemia, ischemia of lung or renal ischaemia prescription face in preparation, and more preferably described polypeptide prevents and/or treats the purposes of myocardial ischemia prescription face in preparation.

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