CN1300171C - Preparation of myocardium peptide - Google Patents

Abstract

The present invention discloses a method for preparing myocardial peptide, which is characterized in that ventricular myocardium which is in a health non-human mammalian is cleaned and minced, and is added with sterile purified water for homogenate; homogenate liquid is repeatedly refrigerated and unfrozen for 3 to 4 times, is heated to 65 to 95 DEG C, and is filtered for removing slag; rough filtration liquid is obtained by that liquid is filtered by a plate frame filter, and is in ultrafiltration by a hollow fibre column; thereby, fine filtration liquid is obtained; ultrafiltration is carried out by an ultrafiltration film, and concentration is carried out by reverse osmosis; thereby, a myocardial peptide solution of which the molecular weight is less than 10000Da is obtained; a finished product is obtained by filtration sterilization and freeze drying. The present invention has the advantages of simple preparation method, moderate product molecular weight, high purity, and high stability; other items are not changed except that the color of appearance is changed into slight yellow when the product is stored for 480 to 540 days.

Description

The preparation method of cardiac muscle peptide element

Technical field

The present invention relates to a kind of preparation method of myocardium peptide element.Specifically, the present invention relates to from the health mammal heart except that human, extract the preparation method of myocardium peptide element, belong to the biochemical technology field.

Background technology

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

Becoming better and approaching perfection day by day and popularizing of cardiac operation for numerous patients have removed misery, improved people's quality of life.Along with people to improving constantly that myocardial preservation requires, the basis is also more and more deep with clinical study.The myocardial preservation of clinical cardiac surgery comprises: reach the postoperative myocardial preservation before the art, in the art, but the emphasis of myocardial preservation still concentrates on the ischemical reperfusion injury aspect of prevention cardiac muscle in the extracorporeal circulation process.To this, basic and clinical scientific worker has carried out deep multi-faceted research, mainly comprise: (1) heart perfusion: mainly concentrate on dabbling mode, for example: along irritate, contrary irritates, irritate simultaneously, be interrupted or continue irritate, whether add and use hemocyte nutsche filter etc.(2) temperature of perfusion liquid: mainly contain normal temperature, low temperature etc.(3) component of perfusion liquid: as add with oxygen free radical scavenger (superoxide-dismutase, reduced glutathion, Trypsin inhibitor,Trasylol, puerarin etc.).More than these measures all improved the pathological change of Myocardial Ischemia Reperfusion Injury to a certain extent.Abroad also have phosphokinase (Neoton, Italy Ou Hui pharmaceutical factory) places perfusion liquid or take trimetazidine (Vasorel, France's Les Laboratoires servier) make cardiac muscle under pathological conditions, improve metabolism, from cell, subcellular structure, oxyradical, energy metabolism, calcium ion (Ca ²⁺) aspect such as excess load set forth some and discovered.But these researchs or emphasis is placed on the supplementing energy aspect isolatedly; or the defense mechanism disengaging of a pre-antisitic defect and body self is come; thereby caused the clinical effectiveness of myocardial preservation unsatisfactory, therefore, research and development more efficient drug protection cardiac muscle is very necessary.

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 still 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.The late nineteen eighties, people observe drosophila larvae through the high thermal treatment of short-term, its salivary gland cell multifibres dyes body " bulk " mode and changes, showing that this regional gene is transcribed is activated, and claim that this is heat-shocked effect (heatshock reaction, HSR, Anna Rev Biochem 1986,55:1151).Find later on laboratory animal in succession through heat-shock pretreatment, can be obviously to the myocardial damage due to the anti-ischemia/reperfusion.Marry was called " ischemic preconditioning " (ischemia precondition with this phenomenon in 1986, IP), discover that further this phenomenon and heat shock induction myocardial cell synthesize one group of new protein-heat shock protein(HSP) (heat shock proteins, HSPs) or claim stress protein (stress protein, SP) relevant.These research reports in succession show that cardiac muscle self has powerful endogenous protection mechanism; Find from prokaryotic organism to the eukaryote again later on, from plant, animal to the people, no matter culturing cell or whole machine body, heat-shocked all has the synthetic expression of HSPs when handling, and show following characteristics: (1) except that heat-shocked can induce HSP synthetic, many factors are still arranged, all can induce HSP synthetic as ischemic, anoxic, ethanol, heavy metallic salt, myocardium pressure load, medicine and most of morbid state, and " cross tolerance phenomenon " can occur; (2) on the HSP structure high conservative is arranged, as fruit bat and yeast HSP ₇₀There is 72% amino-acid sequence identical, human HSP ₇₀Gene and fruit bat HSP ₇₀Gene has 73% homology, HSP ₉₀78% homology is arranged.These are structural similar, guaranteed the homogeny on the function (Burdon:Biochem, J.1986,240:313); (3) HSP has the Best Times scope to myocardial protective effect, claims " window(s) of opportunity " (window ofopportunity) again, exceeds certain time limit just to lose its provide protection (Perdriget:Curr Surg 1989,23); (4) HSP is present in whole organic sphere, is present in the various cells of higher animal body.Inducing of HSPs not only strengthens the myocardial function recovery, and also strengthens the recovery of myocardium endothelial function, prolongs the cardiac arrest time.Above-mentioned discovery is applied to the donor protection of heart transplantation, the treatment of ischemic myocardium, the preparation of extracorporeal circulation cardioplegic solution etc.; can break through the conventional medicament constraint; from strengthening the anti-damage of inductor inner cell potential itself, provide a kind of new way with milestone significance.

People such as Pennica (1995) clone cardiotrophin-1 (CT-1) gene in succession in the myocardial cell, and express in E scherichia coli.Studies show that CT-1 is a kind of cytokine with immunomodulatory and induced growth effect, its natural being present among the myocardial cell, can making the myocardial cell to anti-hypoxia, pyritous infringement, and have the effect that suppresses apoptosis of cardiac muscle.Deep discovers, CT-1 is in cardiac muscle cells and in vivo can induce HSP to express, and this inducing action is and a kind of gp of being called ¹³⁰The cell surface polypeptide relevant, it activates gp ¹³⁰After, make HSP by NF-IL-6/NF-IL--6 β and tyrosine kinase pathway ₇₀, HSP ₉₀The expression that reaches the HSP small-molecule substance is strengthened, and tolerates anoxic and pyritous ability thereby strengthen the myocardial cell.Research shows that also CT-1 can promote the synthetic of myocardial cell's structural protein, and the major axis that increases cell makes contraction stronger.The research of the Myotrophin of gene recombination is another problem of myocardial cell's stimulating factor.Parames (1997) proves that Myotrophin promotes that myocardial cell's growth is relevant with protein kinase-c.Myotrophin and Cardiotrophin may be functional similarities among the myocardial cell, but the different cytokine of activating cells growth pathway has all shown the protection myocardial cell, promote the function of growth.

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, be that preparation is extracted from the heart of healthy young mammals, stable in the pH2-9 scope: as not change in 95-100 ℃ of 10 minutes .60-70 ℃ of 30 minutes following biological activity of heating; 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.

But above-mentioned patented technology has just been carried out separation, purification and simple active testing roughly to this biologically active peptides, and its concrete composition, purposes and effect are not described in detail.

Summary of the invention

The object of the invention is to provide a kind of preparation technology of myocardium peptide element of improvement, and described preparation technology is simple, molecular weight product is moderate, purity is high, good stability, and except that the outward appearance color and luster became little yellow, other project did not have change when storing 480-540 days.

The invention provides a kind of preparation method of myocardium peptide element, the health mammal ventricular muscles that it is characterized in that not comprising the people is cleaned, chopping, adds sterile purified water homogenate, and homogenate is freezing repeatedly, thaw 3～4 times, be heated to 65～95 ℃ of filter cleaners, obtain coarse filtration liquid with the filtration of sheet frame filter, use hollow fiber column ultrafilter again, obtain smart filtrate, use the ultra-filtration membrane ultrafiltration, hold back the myocardium peptide cellulose solution of weight-average molecular weight less than 10000Da, degerming after filtration at last, lyophilize gets finished product.

Healthy Mammals of the present invention is not for to comprise that people's healthy Mammals comprises pig, ox, sheep, rabbit, horse etc., and preferred young mammals comprises sucking pig, cow, milk goat, newborn rabbit, newborn horse etc., most preferably sucking pig.

The add-on of wherein said sterile purified water is 0.5～4 times of mammiferous ventricular muscles; The rotating speed of described homogenate is 1000～5000rpm/min;

Described freezing for being lower than under-5 ℃ the temperature freezing 24～72 hours, preferably-20 ℃～-30 ℃ freezing 36～48 hours down; Described type of heating is for adopting water proof heating or direct heating, and temperature is 70～90 ℃, and the time is preferably adopted the water proof heating for being no more than 2 hours, and temperature is 75 ℃～80 ℃, and the time is for being no more than 1 hour.

The present invention filters with the sheet frame filter and obtains coarse filtration liquid, with obtaining the smart filtrate of molecular weight less than 12kDa behind the hollow fiber column ultrafilter; Dam with the ultra-filtration membrane ultrafiltration again and obtain the smart filtrate of molecular weight less than 10kDa; Wherein said sheet frame filter belongs to the bio-pharmaceuticals conventional equipment, selects for use less than 10 μ middling speed filter paper, preferably is less than or equal to 5 μ middling speed filter paper, and the model of producing such as Guangzhou medicine instrument institute is the sheet frame filter of XAS03-172/8; It is F60 that hollow fiber column can be selected specification for use, can filter the liquid of molecular weight less than 12kDa, such as the hollow fiber column of Switzerland Zinpro Corp.; The ultra-filtration membrane specification is 5-10kDa, and is concentrated with the reverse osmosis concentration post of Millipore company such as the product of Millipore company.

The present invention can be to the myocardium peptide cellulose solution process quality inspection of holding back, and first can as lyophilize is last, described filtration sterilization and can are known in those skilled in the art.

Freeze drying equipment commonly used is adopted in lyophilize of the present invention: freeze drier; Detailed process is: made the interior shelf temperature of kiln reach-15～-20 ℃ in 5～40 minutes, and reached-18～-20 ℃ in preferred 20～30 minutes, again through 20～40 minutes, make products temperature reach-25～-35 ℃, reach-30～-35 ℃ in preferred 25～35 minutes.Keep temperature in the condenser being dropped to-40～-50 ℃ in 1～3 hour, vacuumize again, when vacuum tightness reaches 90～100KPa, be communicated with kiln and condenser, stop the refrigeration of loft drier, when being 10～15Pa, the vacuum tightness of loft drier begins to heat up, heat-up rate is 2～5 ℃/min, is warming up to 5～15 ℃, is incubated 3～6 hours, preferably be warmed up to 8～12 ℃, continue 4～5 hours with 3～4 ℃/min speed.Continuation is warming up to 15～25 ℃ with 8～16 ℃/min speed, continues 3～8 hours, preferably is warmed up to 18～22 ℃ with 10～12 ℃/min speed, continues 4～6 hours.Continuation is warming up to 30～35 ℃ with 7～15 ℃/min speed, continues 1～4 hour, preferably is warmed up to 33～35 ℃ with 9～12 ℃/min speed, continues 1.5～2 hours.Continuation is warming up to 50～60 ℃ with 4～8 ℃/min speed, continues 1～3 hour, preferably is warmed up to 54～58 ℃ with 5～7 ℃/min speed, continues 1.5～2 hours.Enter temperature-fall period, make temperature reduce to 40-50 ℃ in 10～30min, continue 8-15 hour, make temperature reduce to 45～48 ℃ in preferred 15～20min, continue 9～12 hours, obtain the plain dried frozen aquatic products of the qualified myocardium peptide of outward appearance, take out goods and seal.

The present invention's cardiac muscle peptide element is never to comprise the polypeptide that extracts in people's the health mammal heart, its content of peptides is 75%～90%, and total free aminoacids is 6%～15%, and rna content is less than 2%, thymus nucleic acid content is less than 7.5%, and weight-average molecular weight is less than 10000 dalton.

The plain preferred weight-average molecular weight of described myocardium peptide is 2000～8000 dalton, and most preferred weight-average molecular weight is 2000～5000 dalton.

Described myocardium peptide element biological activity in the pH3-8 scope is stable, and to the Proteinase K sensitivity, 85 ℃ of 10min biological activitys do not change, and is stable under freezing or lyophilisation condition.

The plain isoelectric focusing electrophoresis of described myocardium peptide shows 2-6 bar colored zone, and preferred isoelectric focusing electrophoresis shows 2 bands, and wherein pI 10.92 is with painted dark person.

Described myocardium peptide element has a stable maximum absorption band at ultra-violet absorption spectrum 190-210nm place, preferably at ultra-violet absorption spectrum 200 ± 2nm place one maximum absorption band person is arranged.

Adopt sulfosalicylic acid method to identify in the described myocardium peptide element of demonstration and do not contain protein.

The plain vigor of myocardium peptide of the present invention is at least 2.2.

Myocardium peptide element of the present invention is analyzed through FPLC, cardiac muscle peptide element mainly contains 5 component peaks, the percentage area adds up to 90%～95% relatively, through the activity inspection, 5 component peaks all can promote former myocyte and the anoxic oxygen supply myocardial cell succinodehydrogenase vigor again that nourishes heart of being commissioned to train, and wherein P1 peak activity is stronger.

Also can comprise vehicle in the myocardium peptide element of the present invention, its weight ratio consists of:

Cardiac muscle peptide element 15～20

Vehicle 100～375,

Be preferably 18～20: 200～375.

Vehicle can be N.F,USP MANNITOL, trehalose, lactose, sucrose or other freeze-drying auxiliary material, is preferably N.F,USP MANNITOL.

In order to remove the thermal source purpose, also can comprise gac in the myocardium peptide element of the present invention, its content is about 0.1%.

Adopt above-mentioned preparation method, filter type through the filtration of sheet frame filter, hollow fiber column ultrafilter and ultra-filtration membrane ultrafiltration, can obtain required for the present invention after the reverse osmosis concentration less than 10000 dalton cardiac muscle peptide element, compare with ZL in the background technology 94102798, working hour is short, and the treatment capacity of product is many, obtains the concentration height of product, active big, be difficult for producing pyrogen.

Owing in preparation process, adopted the essence filter mode of hollow fiber column ultrafilter and ultra-filtration membrane ultrafiltration, removed high molecular weight protein, therefore be difficult for producing anaphylaxis, cardiac muscle peptide element can directly act on the myocardial cell, promote the reparation that cardiac muscle damages under multiple impairment factor, as ischemic, drug intoxication etc.; Promote protein synthesis, reduce oxygen free radical injury, reduce calcium overload, induce endogenous protection, improve the medicine of myocardial metabolism function,, promote the reparation of damage that a new approach is provided for alleviating induced myocardial injury in the heart operation.

Adopt preparation method's gained cardiac muscle peptide element of the present invention to compare with patent ZL94102798 and the disclosed CMGSP of ZL94102799 (GMGSP), has obviously high external biological activity, the 3-5 that the plain activity unit of myocardium peptide of the present invention is a CMGSP doubly, the drug effect correlation data shows in the body, and it has obvious favourable influence to myocardium creatine phosphokinase release due to myocardial ischemia-reperfusion injury and lactate dehydrogenase activity and free fatty acids and mda content.

The main pharmacodynamics result of study of cardiac muscle peptide element is as follows:

1. myocardium peptide element can obviously alleviate the myocardial ultrastructure damage due to the perfusion of myocardial ischemia-again, make its near or recover normal (Fig. 6-12 black-and-white photograph, table 13).

2. the visceral pericardium electrocardiogram(ECG shows that the ST section that myocardium peptide element can obviously resist due to the cat myocardial ischemia raises, and reduces myocardial ischemia scope (table 14-15).

3. myocardium peptide element can obviously lower increase (the table 16-21) of myocardium creatine phosphokinase release and lactate dehydrogenase activity and free fatty acids and mda content due to myocardial ischemia-reperfusion injury.

4. myocardium peptide element can reduce myocardial consumption of oxygen (table 22).

5. myocardium peptide element 5,10mg/kg can obviously reduce the Electrocardiographic ST of myocardial infarction Pigs Hearts adventitia, reduce NST, and dwindle myocardial infarct size, dead due to quivering in irregular pulse, the chamber that the acute myocardial ischemia pig is occurred have certain therapeutic action, and blood pressure, heart rate are not had obvious influence (table 23, Figure 13).

Description of drawings

The plain HPLC molecular weight of Fig. 1 cardiac muscle peptide collection of illustrative plates

The plain FPLC separation and purification of Fig. 2 cardiac muscle peptide collection of illustrative plates

The plain isoelectric point determination collection of illustrative plates of Fig. 3 cardiac muscle peptide

The plain UV scanning collection of illustrative plates of Fig. 4 cardiac muscle peptide

The plain HPLC of Fig. 5 cardiac muscle peptide differentiates collection of illustrative plates

Fig. 6 cardiac muscle peptide element is to the normal control group of the myocardial ultrastructure damage influence experiment due to the Ischemia-reperfusion Injury

Fig. 7 cardiac muscle peptide element is to the physiological saline control group of the myocardial ultrastructure damage influence experiment due to the Ischemia-reperfusion Injury

Fig. 8 cardiac muscle peptide element is to the control group that damages of the myocardial ultrastructure damage influence experiment due to the Ischemia-reperfusion Injury

Fig. 9 cardiac muscle peptide element is to the plain 10mg/kg group of myocardium peptide of the myocardial ultrastructure damage influence experiment due to the Ischemia-reperfusion Injury, and the myocardial ultrastructure that can obviously alleviate due to the perfusion of myocardial ischemia-is again damaged

Figure 10 cardiac muscle peptide element is to the plain 5mg/kg group of myocardium peptide of the myocardial ultrastructure damage influence experiment due to the Ischemia-reperfusion Injury, and the myocardial ultrastructure that can alleviate due to the perfusion of myocardial ischemia-is again damaged

Figure 11 cardiac muscle peptide element is organized the plain 1mg/kg of myocardium peptide of the myocardial ultrastructure damage influence experiment due to the Ischemia-reperfusion Injury

Figure 12 cardiac muscle peptide element is organized you 2mg/kg of general Lip river naphthalene of the myocardial ultrastructure damage influence experiment due to the Ischemia-reperfusion Injury

Figure 13 cardiac muscle peptide element 5,10mg/kg can obviously reduce the Electrocardiographic ST of myocardial infarction Pigs Hearts adventitia, reduce NST

The plain product process flow figure of Figure 14 cardiac muscle peptide

Embodiment

Following embodiment further describes the present invention, but described embodiment only is used to illustrate the present invention rather than restriction the present invention.

Embodiment 1

This experimental example relates to physico-chemical property, purity, content and the activity experiment of myocardium peptide cellulose solution

One. the physico-chemical property of myocardium peptide element, purity, content

The myocardium peptide that adopts preparation method of the present invention to obtain is plain to be micromolecule active polypeptide, and biological activity is stable in the pH3-8 scope, and to the Proteinase K sensitivity, 85 ℃ of 10min biological activitys do not change, stable under freezing or lyophilisation condition; Analyze through HPLC, weight-average molecular weight is less than 10000Da (Fig. 1), preferable weight-average molecular weight 2000-8000Da.

Under the effect of table 1 condition of different pH, different time to the plain active red land measure equal to fifteen mu in most parts of the Northeast (mtt assay) of myocardium peptide (x ± s, n=8)

Grouping	The plain active OD value of cardiac muscle peptide (x ± s)
				30μg/ml		5μg/ml
	Normal control group Zorubicin damage group	0.691±0.032 **0.274±0.011
Different pH treatment group 3.0 30min 60min 4.0 30min					0.331±0.014 ** 0.314±0.007 ** 0.315±0.008 **	0.320±0.015 ** 0.309±0.010 ** 0.307±0.015 **

5.0 6.0 7.0 8.0 9.0

60min 30min 60min 30min 60min 30mim 60min 30min 60min 30min 60min

0.334±0.015 ** 0.364±0.022 ** 0.364±0.017 ** 0.341±0.023 ** 0.332±0.015 ** 0.320±0.018 ** 0.327±0.010 ** 0.339±0.008 ** 0.308±0.01 **5 0.313±0.006 ** 0.279±0.017

0.311±0.007 ** 0.379±0.019 ** 0.353±0.023 ** 0.344±0.011 ** 0.327±0.016 ** 0.358±0.023 ** 0.328±0.012 ** 0.332±0.022 ** 0.309±0.010 ** 0.289±0.020 0.274±0.013

Annotate: ^*Compare P＜0.01 with the damage group

Can find out in the plain biological activity of pH3-8 scope heart carnosine stable from table 1.

Analyze through FPLC, myocardium peptide element mainly contains 5 component peaks, and the percentage area adds up to 90%-95% relatively.The active inspection, 5 component peaks all can promote former myocyte and the anoxic oxygen supply myocardial cell succinodehydrogenase vigor (table 2) again that nourishes heart of being commissioned to train, wherein P1 peak activity (Fig. 2) by force.The plain content of peptides of cardiac muscle peptide accounts for 75-90%, and total free aminoacids accounts for 6-15%, and trace dna and trace element are still arranged.Isoelectric focusing electrophoresis shows 2 colored zones, and wherein pI 10.92 is with painted dark (Fig. 3).

Plain each component of the myocardium peptide of table 2 is to the influence (mtt assay) of myocardial cell's enzyme activity (n=8, x ± s)

Grouping	The OD value (x ± s)
			5μg/ml	The t value
	Normal control group Zorubicin damage group	0.344±0.014 ** 0.272±0.015			9.93
The plain group of P1 P2 P3 P4 P5 cardiac muscle peptide			0.318±0.004 ** 0.295±0.012 ** 0.309±0.012 ** 0.317±0.017 ** 0.303±0.014 ** 0.298±0.005 **	6.344 3.39 5.45 5.61 4.27 3.47

Annotate: ^*Compare P＜0.01 with the Zorubicin group

5 component peaks all can promote the former myocyte's succinodehydrogenase vigor that nourishes heart of being commissioned to train of Zorubicin damage as can be seen from Table 2.

1. the discriminating of polypeptide: the carnosine cellulose content of coring is the myocardium peptide cellulose solution 1ml of 2.5mg/ml, adds water 2ml dissolving, adds biuret reagent 2ml[biuret reagent preparation method: get copper sulfate (CuSO ₄5H ₂O) 0.75g and Seignette salt (NaKC ₄H ₄O ₆4H ₂O) 3g adds the about 250ml dissolving of water, under agitation add 10% sodium hydroxide test solution 150ml, and thin up is stored in the Plastic Bottle to 500ml.], mixing shows blue purple, is and contains polypeptide; Through 6 batches of inspections, myocardium peptide element of the present invention all shows blue purple, promptly contains polypeptide.

2. assay

(1) semimicro Kjeldahl determination

Cardiac muscle peptide cellulose solution. lot number 960419,960422,960423; Injection cardiac muscle peptide element, 960501,960502.960503, face the time spent water and be dissolved to desired concn.

Reagent of sulfuric acid: chemical pure, proportion 1.84; Digestive pharmaceutical: 1 part in copper sulfate (CuSO4.5H2O) and 10 parts of common porphyrize mixings of vitriolate of tartar (K2SO4); 12.5mol/L sodium hydroxide solution; 2% boric acid absorption liquid; 10% sodium wolframate; 0.33mmol/L sulfuric acid; Mixture indicator: 5 parts of 0.2% (W/V) tetrabromo-mcresolsulfonphthalein spirituous solutions mix for 2 parts with 0.1% (W/V) methyl red spirituous solution and are made into; 0.01mol/L hydrochloric acid.

Calculation formula:

Assay method is measured according to N2 method (seeing two appendix VII of Chinese Pharmacopoeia nineteen ninety-five version D, second method).

Inorganic nitrogen: precision is measured trial-product 5ml, adds water 3ml, adds 10% sodium wolframate 1ml, and 0.33mmol/L sulfuric acid 1ml shakes up, and leaves standstill 30 minutes, filters.Precision is measured filtered liquid 5ml and sodium hydroxide test solution 5ml, adds in the matrass, shines under the total nitrogen item with the method determination of distillation.

Total nitrogen: get plain one bottle of injection cardiac muscle peptide, accurately add water 4ml dissolving, precision is measured 2.0ml; Precision is measured myocardium peptide cellulose solution 2.0ml, measures according to the determination of total nitrogen content method respectively.

Organonitrogen amount=total nitrogen-inorganic nitrogen amount

Annotate: (1) is when measuring inorganic nitrogen, because trial-product is easy to generate foam in still-process, bring sodium hydroxide into prolong, flows into and collect in the liquid, make measurement result higher, should before distillation, add 10% sodium wolframate and 0.33mmol/L sulfuric acid and remove organism. get filtrate and measure inorganic nitrogen.

(2) trial-product is major ingredient with the peptide material, influences measurement result owing to bringing inorganics generation inorganic nitrogen in process of production into.This law is measured inorganic nitrogen after measuring total nitrogen, deduct the inorganic nitrogen amount as the organonitrogen amount with total nitrogen.

Result and analysis

The nitrogen content (seeing Table 3) of different lot number cardiac muscle peptide cellulose solutions and preparation.

The different lot number trial-product of table 3. nitrogen determination result

	Cardiac muscle peptide cellulose solution (mg nitrogen/ml)			Injection cardiac muscle peptide element (mg nitrogen/bottle)
							Lot number	960419	960422	960423	960501	960502	960503
Total nitrogen	1.788	1.926	1.628	3.816	4.250	4.100
							Organonitrogen	1.589	1.743	1.460	3.612	3.919	3.722
Inorganic nitrogen	0.199	0.183	0.168	0.204	0.331	0.378

Table 3 shows that myocardium peptide cellulose solution organonitrogen content is between 1.46-1.74mg/ml, and injection cardiac muscle peptide have the machine nitrogen content in 3.61-3.92mg/ bottle scope.Average content is respectively 1.60mg nitrogen/ml and 3.75mg nitrogen/bottle.

(2) forint--phenol method (Folin-phenol)

Cardiac muscle peptide cellulose solution, lot number 960419,960422,960423; Injection is the peptide element diligently, lot number 960501,960502,960503; Face the time spent water and be dissolved into suitable concentration.Reference substance: bovine serum albumin (Nat'l Pharmaceutical ﹠ Biological Products Control Institute) lot number 9607.

Instrument 7221 type spectrophotometers, Shanghai.

Reagent preparation: 4% sodium carbonate solution is got yellow soda ash (Na2CO310H2O) 4g and is dissolved in water and is diluted to 100ml, shakes up.0.2mol/L sodium hydroxide solution is got sodium hydroxide (NaOH) 0.8g, is dissolved in water and dilutes 100ml, shakes up.1% copper-bath is got copper sulfate (CuSo45H2O) 1g and is dissolved in water and is diluted to 100ml, shakes up, promptly.2% soluble tartrate solution is got soluble tartrate (K2C4H4O61/2H2O) 2g, is dissolved in water and is diluted to 100ml, shakes up.

The alkaline copper test solution faces with before getting test solution 1 and 2 each 25ml, test solution 3 and 4 each 0.5ml, mixing.

The phenol test solution is got sodium wolframate (Na2WO42H2O) 100g, Sodium orthomolybdate (Na2MoO4.2H2O) 25g, put in the 1500ml flask, add water 700ml, 85% phosphoric acid 50ml, hydrochloric acid 100ml, on connect little the boiling of return line (with the soft rubber ball of cork stopper or tinfoil parcel) and refluxed 10 hours, take off prolong, add Lithium Sulphate (Li ₂SO ₄) 150g, complete molten after, add water 50ml and bromine liquid number droplet again, shake up, boiled 15 minutes, drive away excessive bromine.Be cooled to room temperature, thin up filters to 1000ml, and filtrate is stock solution, stores in the brown bottle, puts in the refrigerator and preserves.Face the time spent, get stock solution and use water as doubly amount dilution, promptly.

Typical curve is drawn

The preparation precision of reference substance solution takes by weighing exsiccant bovine serum albumin reference substance (Nat'l Pharmaceutical ﹠ Biological Products Control Institute provides) 100mg, puts in the 100ml measuring bottle, and thin up is to scale, shake up, precision is measured 10.0ml, puts in the 100ml measuring bottle, thin up shakes up promptly to scale.

The preparation precision of typical curve is measured reference substance solution 0.0,0.2,0.4,0.6,0.8 and 1.0ml, put respectively in the tool plug test tube, and each Guan Jiashui makes into 1.0ml, and every pipe adds alkaline copper solution 5.0ml, shakes up, and room temperature was placed 10 minutes.Add phenol reagent 0.5ml more successively fast, shake up immediately, put in the water-bath (35 ℃) and be incubated 30 minutes.Take out, be cooled to room temperature, make blank, measure optical density at 660nm wavelength place according to spectrophotometry (two appendix IV of Chinese Pharmacopoeia nineteen ninety-five version A) with reference substance solution 0.0ml pipe.With the optical density is ordinate, and protein concn is an abscissa, the drawing standard curve.

Assay method is got this product, is dissolved in water respectively or dilutes and quantitatively be transferred in the 50ml measuring bottle, adds water to scale, shakes up.The accurate 10.0ml that draws puts in the 50ml measuring bottle, adds water to scale, shakes up.The accurate 1.0ml that draws puts in the tool plug test tube, plays the mensuration optical density from " adding the alkaline copper test solution " under the sighting target directrix curve preparation, checks in respective concentration on typical curve, calculates, promptly.

Result and analysis

Measured result sees Table 4.

The every 1ml of measured result cardiac muscle peptide cellulose solution contains polypeptide in the 2.6-2.8mg scope; Injection cardiac muscle peptide contains 9.2-9.9mg for plain every bottle.Constant for guaranteeing solution and formulation content, we stipulate that myocardium peptide cellulose solution content of peptides should inject the plain every bottle of content of peptides of myocardium peptide at 9.0-11.0mg greater than every milliliter of 2.5mg.

The measurement result of three kinds of content assaying methods of table 4 relatively

	Biuret method	The measuring method forint-phenol law	Nitriding (polypeptide organonitrogen)
				Solution (mg/ml)
960419 960422 960423	1.65 1.85 2.00	2.7 2.8 2.6	1.58 1.74 1.46
				Preparation (mg/ bottle)
960501 960502 960503	3.26 3.63 3.73	9.9 9.2 9.2	3.61 3.92 3.72

Annotate: ^*Biuret method is measured by automatic clinical chemistry analyzer

Three kinds of measuring methods are inconsistent as can be seen from Table 10, and the result is quite different.The reaction principle of considering forint-phenol law is the phenolic group reaction of die aromatischen Aminosaeuren, and specificity is better, and is easy and simple to handle.Select for use specific reference substance and the equal drawing standard curve of each mensuration can overcome nonlinear relationship, we select for use forint-phenol law to measure the content of peptides of myocardium peptide cellulose solution and injection cardiac muscle peptide element.

(3) proportion of composing analysis

These goods mainly are made up of polypeptide, measure its organonitrogen content respectively, deduct total free aminoacids organonitrogen content by total organic nitrogen content and are polypeptide organonitrogen content.

3.1 reagent and method the same (seeing nitriding).

3.2 result

3.2.1 the plain total free aminoacids nitrogen content of the injection of 3 lot numbers cardiac muscle peptide sees Table 5.

The plain total free aminoacids nitrogen content of table 5. injection cardiac muscle peptide

	Injection cardiac muscle peptide element
				Lot number	960501	960502	960503
Total free aminoacids total nitrogen g/L	0.257	0.285	0.286

3.2.2 injection cardiac muscle peptide have the machine nitrogen content and the total free aminoacids nitrogen content compares (seeing Table 6)

Table 6. total nitrogen and total free aminoacids nitrogen content are relatively

	Injection cardiac muscle peptide element
				Lot number	960501	960502	960503
Polypeptide organonitrogen g/L	3.612	3.919	3.722
				Free Amino Nitrogen	0.257	0.285	0.286
The total free aminoacids nitrogen content accounts for total organic nitrogen amount per-cent (%)	6.643	6.823	7.135

From table 5,6 as can be seen, and the plain total free aminoacids nitrogen content of injection cardiac muscle peptide only accounts for the 6.643%-7.135% of trial-product nitrogen content, shows that the trial-product polypeptide accounts for the overwhelming majority of total nitrogen.In view of this product polypeptide is the major ingredient of biologic activity and the stable and controllable of considering production technique, the plain polypeptide ratio of our regulation injection cardiac muscle peptides should reach 75%-90%.

3. UV scanning analysis:

Day island proper Tianjin 2201 type ultraviolet spectrophotometers are measured according to spectrophotometry (two appendix IV of Chinese Pharmacopoeia nineteen ninety-five version A).

The result shows that myocardium peptide cellulose solution has maximum absorption band at the 199.8-201.2nm place, and injection cardiac muscle peptide element shows maximum absorption band (Fig. 4) at the 200.4-201.8nm place.Show that 3 batches of solution are consistent with 3 batches of preparation UV scanning collection of illustrative plates, the trial-product main component is a polypeptide, and the plain reparation technology of myocardium peptide is stable.

The UV scanning absorbing wavelength of the myocardium peptide element of table 7

Lot number	Absorbing wavelength (nm)
		Solution, (mg/ml) 960,422 960,423 960419 preparations, (mg/ bottle) 960,501 960,502 960503	200.4 199.8 201.2 200.4 201.8 201.6

4. proteinic discriminating: get the plain 2ml of myocardium peptide of the present invention's cardiac muscle peptide cellulose content 2.5mg/ml, add 20% sulphosalicylic acid solution 1ml, do not produce muddiness, plain three batches of solution of myocardium peptide and the demonstration of preparation measurement result all do not contain protein.Check protein with sulfosalicylic acid method, both can monitor that the shown blue purple of also provable biuret method is polypeptide rather than other material simultaneously to the protein that may sneak into.

5. molecular weight and peptide color atlas are checked: HPLC method determining molecular weight

The HP1050 liquid chromatograph

Chromatographic condition: moving phase: sodium sulfate (0.1mol/L)-SODIUM PHOSPHATE, MONOBASIC (0.05mol/L)-sodium azide (0.05%), transfer pH to 6.8 with NaOH; Flow velocity: 0.35ml/min; Post: TOSOH TSK G2000sw 7.5mm * 300mm; Column temperature: 25 ℃; Detect wavelength: 280nm; Sample size: 10 μ l.

Get cytochrome C (MW=12400) respectively, Trypsin inhibitor,Trasylol (MW=6700) and vitamins B ₁₂(MW=1355) in right amount, make the reference substance solution of suitable concentration respectively with moving phase.Get plain 1 bottle of trial-product injection cardiac muscle peptide, content of peptides 10mg makes the need testing solution that every 1ml contains 5mg with moving phase.Get reference substance solution and need testing solution respectively, press the chromatographic condition injecting chromatograph, measure its retention time respectively.Compare the regression equation of product with method of least squares, relation conefficient must not be less than 0.99.Press following formula drawing standard curve and calculate the trial-product molecular weight.

IgMW＝A+BtR

IgMW＝6.8405-0.1219tR γ＝-0.9990

MW is a molecular weight in the formula, and A is a constant, and B is a slope, tR be retention time (minute).

The results are shown in Table 8,9.

The retention time of the relative percentage area of the plain chromatographic peak of table 8 injection cardiac muscle peptide

Lot number	Chromatographic peak retention time (min)
						P1	P2	P3	P4	P5
	960501	30.5	31.8	32.8	35.0						38.7
960502						30.5	31.2	32.8	35.0	38.7
	960503	31.2		32.7	35.0						38.7
X						30.7	31.5	32.7	35.0	38.7

The peak molecular weight of table 9 injection cardiac muscle peptide element

Lot number	Molecular weight (dalton)
						P1	P2	P3	P4	P5
	960501	6023	4588	3665	2233						969
960502						6027	5261	3688	2234	971
	960503	5165	3709		2236						875
X						5736	4519	3676	2234	971

X＝6.7444-0.09696×Y＝-0.9937

The range of molecular weight distributions of injection cardiac muscle peptide element is at 922-6027Da, and the maximum molecular weight scope shows that at 5214-6027Da (Fig. 1) this trial-product is a micromolecule polypeptide, and molecular weight is difficult for producing anaphylaxis less than 10000 dalton.

6. nucleic acid: injection cardiac muscle peptide element, every bottle contains polypeptide 10mg.Adding distil water 4ml dissolving adds the extracting of equal-volume phenol once again.Get supernatant and measure dna content.With resetting and adding the 10mol/L ammonium acetate of long-pending no water-cooled ethanol of amphiploid and 1/20 volume on this, put-80 ℃, 30min, 12000rpm * 20min abandons supernatant, and precipitation is dissolved in the 4ml distilled water.This sample is used to measure rna content.

(1) rna content is measured

The preparation of typical curve:

Get 6 in test tube, according to the form below adds reagent

	Add-on ml
							Admixture
	0	1	2	3	4	5
							The RNA reference liquid	0	0.2	0.4	0.6	0.8	1.0
Distilled water	1.0	0.8	0.6	0.4	0.2	0
							Orcinol reagent	3.0	3.0	3.0	3.0	3.0	3.0

With each pipe mixing, put and heat 20min in the burning water, take out, cold water is cooled to room temperature,, with " 0 " number pipe zeroing, measures and respectively manages absorbancy under the 670nm wavelength with spectrophotometer.With the rna content is X-coordinate, and absorbancy is an ordinate zou, the drawing standard curve.

The sample rna content is measured:

Get 4 in test tube, be marked with " blank pipe " and " measuring pipe " respectively.In blank pipe, add distilled water 1.0ml, in measuring pipe, add RNA sample solution 1.0ml, every then pipe adds orcinol reagent 3.0ml, and mixing is put 20min in the boiling water, take out, cooling bath is cooled to room temperature,, returns to zero with the blank pipe under the 670nm wavelength with spectrophotometer, working sample pipe absorbancy is found rna content and is averaged on typical curve.

(2) dna content is measured

The preparation of typical curve:

Get 6 in test tube, according to the form below adds reagent

	Add-on ml
							Admixture
	0	1	2	3	4	5
							The RNA reference liquid	0	0.2	0.4	0.6	0.8	1.0
DH 2O	1.0	0.8	0.6	0.4	0.2	0
							Pentanoic reagent	3.0	3.0	3.0	3.0	3.0	3.0

With each pipe mixing, put in 60 ℃ of water-baths and heat 60min, take out, cold water is cooled to room temperature, uses spectrophotometer, under the 595nm wavelength, with " 0 " number pipe zeroing, measures and respectively manages absorbancy.With the dna content is X-coordinate, is ordinate zou with the absorbancy, the drawing standard curve.

The sample DNA assay:

Get 3 in test tube, be marked with " blank pipe " and " measuring pipe " (3) respectively, in blank pipe, add distilled water 1.0ml, in measuring pipe, add DNA sample solution 1.0ml, every then pipe adds pentanoic reagent 3.0ml, mixing is put heating 60min taking-up in 60 ℃ of water-baths, puts cooling bath and is cooled to room temperature.Under the 595nm wavelength, return to zero working sample pipe absorbancy with the blank pipe with spectrophotometer.On typical curve, find dna content, average.

The result shows: injection cardiac muscle peptide element contains RNA for every bottle and is no more than 200 μ g (2%); DNA is no more than 750 μ g (7.5%).

7. vigor

Adopt former generation myocardial cell culture method.

Be subjected to the reagent thing: the plain lot number of injection cardiac muscle peptide is 960501,960502,960503,960101, and content of peptides is the 10mg/ bottle.

Experimental result: (the results are shown in Table 10).

The plain vitality test t value (n=6) of the myocardium peptide of table 10

Lot number	The t value
		960501 960502 960503 960101	5.8 3.2 7.9 7.8

8.HPLC method is differentiated injection cardiac muscle peptide element

Instrument: HP1100, Module liquid chromatograph No DE 70300954

Chromatographic condition: moving phase: methyl alcohol: water=10: 90

Post: ymc-park ODS-A A-302 150mm * 4.6mm I.D S-5 μ m 120A No041543847 (W)

Column temperature: 26 ℃.Detect wavelength: 254nm.Flow velocity: 0.8ml/min.Sample size: 10 μ l.

Assay method: get trial-product, every bottle adds moving phase 10ml, after treating to dissolve fully, is for experiment.

The trial-product lot number is respectively 960101,960501,960502,960503,961101,961103,971201,980301.

The result shows: 10 batches of trial-products mainly show 4-5 main peak, and relative percentage peak area is greater than 85%, each main peak retention time very close (seeing Fig. 7, table 11,12).

Each main peak retention time of table 11

	Retention time (min)
										Main peak	101	501	502	503	1101	1102	1103	201	301
P1 P2 P3 P4 P5	1.922 2.506 2.654 3.156 4.240	1.901 2.504 2.651 3.144 4.203	1.920 2.500 2.646 3.134 4.181	1.915 2.502 2.645 3.128 4.159	1.925 2.643 3.124 4.148	1.924 2.639 3.114 4.128	1.949 2.640 3.117 4.133	1.954 2.638 3.115 4.129	1.990 2.638 3.115 4.107

The relative percentage peak area of each main peak of table 12

	Percentage area %
										Main peak	101	501	502	503	1101	1102	1103	201	301
P1 P2 P3 P4 P5	18.3 10.6 10.4 37.4 13.2	12.3 11.7 13.3 45.4 8.8	13.4 9.2 11.1 47.8 9.6	15.7 12.4 8.8 38.6 15.9	13.4 5.2 43.8 22.8	13.5 5.2 43.4 22.6	11.4 5.1 47.3 22.9	14.4 5.8 46.8 18.5	16.1 4.7 42.2 23.6

4 ℃ of trial-products of storing 3 years to 11 months are analyzed at aforementioned chromatographic condition, the retention time of the trial-product of 10 lot numbers is very approaching, and the ratio (the relative percentage area addition at 3 peaks is greater than 66%) of selecting main peak 1,4,5 relative retention times is as index of discrimination.Main peak 1,4,5 relative retention time ratios should be 1: 1.61: 2.14 (± 0.1) as calculated.

Embodiment 2

The present invention has carried out the test of main pharmacodynamics to described myocardium peptide element, has observed myocardium peptide element to the influence of myocardium morphological indexes, physical signs and biochemical indicator and to the influence of myocardial consumption of oxygen on whole and isolated myocardium ischemic and ischemia-reperfusion model.Result of study is as follows:

1. the influence that myocardium peptide element damages the myocardial ultrastructure due to the ischemia-reperfusion

The reference method, behind the ligation rat coronary artery LAD 5min, sublingual vein is injected the plain or contrast medicine of myocardium peptide, unclamps ligature behind the myocardial ischemia 10min, irritates 30min again, writes down the II ECG that leads simultaneously.Irritate to finish the back again from abdominal aortic blood, take out heart, after aorta perfusion is clean towards Xian, with 6% glutaraldehyde 0.1M sodium cacodylate buffer liquid perfusion and fixing 2h, get left front wall ischemic myocardium again, be cut into 1mm with physiological saline ³Fritter immerse in the 4% glutaraldehyde 0.1M sodium cacodylate buffer liquid fixing, in order to making electron microscope specimen.After osmic acid was fixing, serial acetone dehydration was cut into slices after 618 Resins, epoxy embeddings and the polymerization, and every animal cuts 4 embedded blocks.20 of every treated animal random picture, the egative film multiplying power is 12000, observes ultrastructural change, presses the pathology kind and the severity classification definite value of plastosome, cardiac muscle fibre and the damage of other composition.Experiment is divided into 7 groups, is respectively sham-operation (P-O) group; Ischemia-reperfusion (I-R) group; Ischemic-filling group again+physiological saline or Propranololum (I-R+N.S, I-R+Pro) group; Ischemia-reperfusion+3 dosage groups of myocardium peptide element (I-R+MTP).

The myocardium peptide element of table 13 is to the histological influence of ischemia-reperfusion rat heart muscle Electronic Speculum sxemiquantitative (n=20, x ± s)

Group	Dosage (mg/Kg)	Pathology value ± SD
			Sham-operation ischemia-reperfusion ischemia-reperfusion+physiological saline ischemia-reperfusion+myocardium peptide ischemia-reperfusion+inderal	-- -- -- 1.0 5.0 10.0 2.0	0.36±0.46 1.97±1.4△△△ 2.68±1.3 * 1.85±1.6 * 0.73±0.96 *** 0.33±0.42 *** 0.71±0.84 ***

Annotate: compare △ △ △ P＜0.01 with Sham-operated control group; Compare with the ischemia-reperfusion group, ^*P＞0.05, ^{* *}P＜0.01

Can find that by test myocardium peptide element can obviously alleviate the myocardial ultrastructure damage due to the perfusion of myocardial ischemia-again, make its near or recover normal (seeing Fig. 6-12).

2. myocardium peptide element is to the influence of myocardial ischemia

The reference method also corrects, cat opens cuts off LAD place pericardium after chest exposes heart, cause acute myocardial ischemia 10min with the plastic casing compression method, unclamp 30min, the cloth sheet that will have 5 groups of (3 every group) electrodes is sewn in ischemic myocardium pericardium place, write down every group of I, II, the III epicardial electrogram of leading, go femoral arteriography record arteriotony simultaneously.Get ischemic 1 ', 4 ', 7 ' and irritate 1 again ', 5 ', 10 ', 20 ' and continue blocking-up 1 ', 5 ', 10 ', 15 ', 20 ', 30 ', 40 ', 50 ', 60 ' be some writing time.Raise or the millivolt number that descends represent ST section change with the ST section.Every cat blocking-up 5 times, the 5min vein is given different medicines before the 4th blocking-up, continues blocking-up LAD the 5th time, and in continuing blocking-up back 20min, 30min, 40min vein give the myocardium peptide element of various dose, and gains in depth of comprehension are satisfied with to continue administration behind the blocking-up 30min.∑ △ ST and ∑ NST when statistics is respectively organized the 3rd, 4,5 blocking-up respectively.Experiment is divided into ischemia-reperfusion group (I-R): ischemia-reperfusion+physiological saline group (I-R+N.S); Ischemia-reperfusion+myocardium peptide element 2.0,5.0, (I-R+MTP 2.0,5.0,10.0mg/kg) for the 10.0mg/kg group; And totally 6 groups of ischemia-reperfusions+Propranololum group 2.0mg/kg (I-R+Pro).

The plain preventive administration of the myocardium peptide of table 14. is to the influence of cat epicardial electrogram ischemic stage ∑ △ ST and ∑ NST (x ± s)

Group	Dosage (mg/Kg)	n	∑△ST (mV)	∑ NST (individual)
					Ischemia-reperfusion group ischemia-reperfusion+physiological saline group ischemia-reperfusion+cardiac muscle is eliminated plain group	-- -- 2.0 5.0	10 10 6 6	109±32 115±24 *70.8±16 ***37.8±12 ***	28.9±5.2 31.1±5.1 *19.5±4.8 ***9.33±3.9 ***

Annotate: compare with the ischemia-reperfusion group, ^*P＞0.05; ^{* *}P＜0.01

The administration of the myocardium peptide extract for treating of table 15. is to the influence of cat epicardial electrogram ∑ △ ST and ∑ NST (x ± s)

Group	Dosage (mg/Kg)	n	∑△ST (mV)	∑ NST (individual)
					Ischemia group ischemic+physiological saline group ischemic+plain the group of myocardium peptide ischemic+Propranololum group	-- -- 2.0 5.0 10.0 2.0	12 12 6 6 6 6	40.5±10 40.0±12 * 29.9±2.9 *** 25.6±5.7 *** 19.7±4.0 *** 22.8±6.4 ***	11.1±2.1 11.2±1.5 * 8.67±2.2 ** 7.33±1.5 *** 6.17±1.2 *** 6.17±1.5 ***

Annotate: compare with the ischemia-reperfusion group, ^*P＞0.05, ^*P＜0.05, ^{* *}P＜0.01

Show that by the visceral pericardium electrocardiogram(ECG ST section that myocardium peptide element can obviously resist due to the cat myocardial ischemia raises, and reduces the myocardial ischemia scope.

3. myocardium peptide element is to the influence of myocardium creatine phosphokinase release due to myocardial ischemia-reperfusion injury and lactate dehydrogenase activity and free fatty acids and mda content

(1) the reference method is made the Myocardial Ischemia Reperfusion Injury animal model, sublingual vein injection MTP or verapamil (verapamil.Ver) back 5min ligation rat coronary artery left anterior descending branch 10min, pour into 30min again, with the ECG that leads that physiograph is observed continuously and record II leads more, get left painstaking effort 2ml after irritating end again, heart is got left ventricular apex portion cardiac muscle behind aorta perfusion, 4C preserves, and detects in the 48h.The experiment grouping: experiment is divided into Sham-operated control group (pseudo-operation.P-O); The ischemia-reperfusion group (ischemia-reperfution, I-R); Ischemia-reperfusion+physiological saline group (I-R+N.S); 7 groups of ischemia-reperfusion+myocardium peptide element 0.5,2.0,10.0mg/Kg group (I-R+MTP) and ischemia-reperfusion+verapamil 1.0mg/Kg (I-R+Ver) etc., every group of 8-10 animal.

The plain preventive administration of the myocardium peptide of table 16. is to ischemia-reperfusion rat heart muscle and the active influence of plasma C PK (X ± s)

Group	Dosage (mg/Kg)	n	Cardiac muscle CPK (u/100mg pro)	Plasma C PK (u/100ml)
					Sham-operation ischemia-reperfusion ischemia-reperfusion+physiological saline ischemia-reperfusion+myocardium peptide ischemia-reperfusion+Verapamil ischemia-reperfusion+CMGSP	0.5 2.0 10.0 1.0 5.0	10 10 8 8 8 8 8 8	980±63 522±65△△△ 501±59 *732±98 ***904±95 ***976±95 ***886±115 ***830±97 ***	164±64 374±54△△△ 337±48 * 210±50 *** 157±31 *** 134±24 *** 192±60 *** 199±35 ***

Annotate: compare △ △ △ P＜0.01 with Sham-operated control group; Compare with the ischemia-reperfusion group, ^*P＞0.05, ^{* *}P＜0.01

The plain preventive administration of the myocardium peptide of table 17. is to ischemia-reperfusion rat heart muscle and the active influence of blood plasma LDH (x ± s)

Group	Dosage (mg/kg)	n	Cardiac muscle LDH (u/mg pro)	Blood plasma LDH (u/ml)
					Sham-operation ischemia-reperfusion ischemia-reperfusion+physiological saline ischemia-reperfusion+myocardium peptide ischemia-reperfusion+Verapamil ischemia-reperfusion+CMGSP	- - - 0.5 2.0 10.0 1.0 5.0	10 10 8 8 8 8 8 8	76.7±19 110±27△△△ 112±19 *97.1±12 *76.3±22 ***64.8±17 ***75.1±23 ***83.0±17 ***	40.9±9.5 120±20△△△ 116.2±12 *93.9±17 ***59.7±12 ***52.6±13 ***46.7±8.8 ***60.9±15 ***

Annotate: compare with Sham-operated control group, compare with the ischemia-reperfusion group △ △ △ P＜0.01, ^*P＞0.05, ^{* *}P＜0.01

The plain preventive administration of the myocardium peptide of table 18. is to the influence of ischemia-reperfusion rat heart muscle and contents of mda (x ± s)

Group	Dosage (mg/kg)	n	Cardiac muscle MDA (nmol/100mg pro)	Blood plasma MDA (nmol/ml)
					Sham-operation		10	68.3±8.4	22.3±1.8

Ischemia-reperfusion ischemia-reperfusion+physiological saline ischemia-reperfusion+myocardium peptide ischemia-reperfusion+Verapamil ischemia-reperfusion+CMGSP

0.5 2.0 10.0 1.0 5.0

10 8 8 8 8 8 8

135±10△△△ 127±15 *73.1±13 ***60.5±10.4 ***49.8±9.4 ***66.6±19.8 ***65.2±9.7 ***

63.6±11△△△ 58.4±11 *38.1±6.2 ***27.7±5.5 ***25.5±5.1 ***24.9±6.6 ***32.2±5.3 ***

Annotate: compare △ △ △ P＜0.01 with Sham-operated control group; Compare with the ischemia-reperfusion group, ^*P＞0.05, ^{* *}P＜0.01

The myocardium peptide element of table 19 is to the influence of ischemia-reperfusion rat blood serum FFA content (n=8, x ± s)

Group	Dosage (mg/Kg)	FFA (μmol/100ml)
			Sham-operation ischemia-reperfusion ischemia-reperfusion+physiological saline ischemia-reperfusion+myocardium peptide ischemia-reperfusion+inderal ischemia-reperfusion+CMGSP	-- -- -- 1.0 5.0 10.0 2.0 5.0	60.6±7.8 129±26△△△ 121±10 * 85.4±5.0 *** 77.7±7.1 *** 71.4±11 *** 77.1±6.4 *** 79.2±6.7 ***

Annotate: compare △ △ △ P＜0.01 with Sham-operated control group; Compare with the ischemia-reperfusion group, ^*P＞0.05, ^{* *}P＜0.01

Adopt preparation method's gained cardiac muscle peptide element of the present invention to compare with patent ZL94102798 and the disclosed CMGSP of ZL94102799 (GMGSP), has obviously high external biological activity, the 3-5 that the plain activity unit of myocardium peptide of the present invention is a CMGSP doubly, the drug effect correlation data shows in the body, and it has obvious favourable influence (seeing Table 16-19) to myocardium creatine phosphokinase release due to myocardial ischemia-reperfusion injury and lactate dehydrogenase activity and free fatty acids and mda content.

(2) the reference method is made isolated rat Langendorffs heart anoxic-reoxygenation injury animal model, with high Ca2+, the K-H liquid that low K+ and continuing charges into mixed gas carries out the Langendorffs perfusion, and two platinum filaments are colluded respectively in the apex of the heart and left room root, recording ecg.LAD ligation 10min unclamps 15min, the administration group before ligation 5min to unclamp the back 5min all to contain the K-H liquid perfusion of respective concentration medicine.8min and the effluent liquid that unclamps back 2min are measured relevant index before collecting ligation respectively, after the ligation.Perfusion is got left chamber front and back walls cardiac muscle after finishing, and 4 ℃ of preservations detect CPK, LDH and MDA in the 48h.The experiment be divided into Sham-operated control group (pseudo-operation, P-O); Anoxic-reoxygenation group (anoxia-reoxygenation, A-R); 6 groups of anoxic-reoxygenation+myocardium peptide element 10,50,100 μ g/ml (final concentration A-R+MTP) group and anoxic-reoxygenation+verapamil 1.0 μ g/Kg (A-R+Ver) etc., every group of 10 animals.

The active influence of coronary artery effluent liquid CPK during to the perfusion of isolated rat myocardial ischemia-again of the myocardium peptide element of table 20. (n=10, x ± s)

Group	Dosage (μ g/ml)	Coronary artery effluent liquid CPK (U/L)
					Before the ischemic	Ischemic stage	Irritate the phase again
		Sham-operation ischemiareperfusion ischemiareperfusion+myocardium peptide ischemiareperfusion+Verapamil	-- -- 10 50 100 1	15.3±1.5 16.3±2.3△ 16.1±2.6 *15.6±1.7 *15.5±2.7 * 16.3±2.0 *				16.5±1.8 24.8±2.7△△△ 20.7±1.7 ***17.9±2.7 ***15.3±2.1 *** 16.2±2.8 ***	17.1±2.0 35.4±4.3△△△ 22.7±2.3 ***19.0±2.3 ***16.5±2.4 *** 16.0±1.8 ***

Annotate: compare △ P＞0.05, △ △ △ P＜0.01 with Sham-operated control group; With ischemic-hemoperfusion group compares again,

^*P＞0.05， ^***P＜0.01

The myocardium peptide element of table 21. is to isolated rat myocardial ischemia-active influence of perfusion coronary artery effluent liquid LDH again (n=10, x ± s)

Group	Dosage (μ g/ml)	Coronary artery effluent liquid LDH (U/L)
					Before the ischemic	Ischemic stage	Irritate the phase again
		Sham-operation ischemiareperfusion ischemiareperfusion+myocardium peptide ischemiareperfusion+Verapamil	-- -- 10 50 100 1	11.8±0.79 11.7±0.83△ 12.0±0.58 *11.8±0.53 *11.2±0.55 * 11.4±0.78 *				12.6±1.1 17.3±1.9△△△ 13.4±1.1 ***12.9±1.1 ***12.2±0.79 *** 13.0±0.62 ***	11.8±0.69 24.7±1.7△△△ 15.3±1.4 ***13.4±0.76 ***12.9±0.93 *** 14.3±0.95 ***

Annotate: compare △ P＞0.05, △ △ △ P＜0.01 with Sham-operated control group; With ischemic-hemoperfusion group compares again,

^*P＞0.05. ^***P＜0.01

Show that by test myocardium peptide element can obviously lower increasing of myocardium creatine phosphokinase release and lactate dehydrogenase activity and free fatty acids and mda content due to myocardial ischemia-reperfusion injury.

4. myocardium peptide element is to the influence of myocardial consumption of oxygen

The anesthesia of dog vetanarcol, trachea cannula, artificial respiration is led physiograph monitoring electrocardiogram(ECG and aortic pressure with the RM-86 type more.Chest is opened in the left side, exposes heart, from apex of the heart intubate to left ventricle, left constant pressure and rate of pressure change (± dp/dt max).For understanding coronary circulation and myocardium O ₂Metabolic variation, the LCA of separation dog is surveyed coronary flow with magnetic flow meter, calculates coronary resistance.Extremely crown from the external jugular vein intubate of dog, extract arterial blood and coronary sinus blood simultaneously, measure blood O with Bloodgas Analyzer (ABL-3 type, Denmark) ₂Content, calculating myocardium O ₂Uptake ratio and cardiac muscle consumption O ₂Amount.The arterial blood ph CO that keeps dog in the experimentation ₂With O ₂Divide and be pressed in normal range.MTP dosage is 2,5,10mg/kg, two spacing of doses 30min.Continuous recording parameters after the administration is until returning to control value substantially.After the administration 2,5,10,30min extracting arterial blood and coronary sinus blood are surveyed vim and vigour content, calculating myocardium O ₂Uptake ratio and cardiac muscle consumption O ₂Amount is observed MTP to myocardium O ₂Metabolic influence.

The myocardium peptide element of the quiet notes of table 22. is to the influence (changing value % after the administration) of dog myocardial consumption of oxygen and myocardium coefficient of oxygen utilization

Time (min)	Myocardial consumption of oxygen (MVO 2)	Cardiac muscle coefficient of oxygen utilization (O 2ext)
				The plain 2mg/kg (N=8) of cardiac muscle peptide
2 5 10 20	-23.0±26 -21.0±13 *** -18.0±14 -9.00±12	-4.00±13 0±8.0 -2.00±6.0 -2.00±12
				The plain 5mg/kg (N=7) of cardiac muscle peptide
2 5 10 20	-36.0±24 **-26.0±21 **-19.0±15 **-8.00±10	-5.00±13 6.00±8.0 6.00±6.0 * -14.0±35
				The plain 10mg/kg (N=6) of cardiac muscle peptide
5 10 20 30	-22.0±25 -21.0±14 **-8.00±4.0 *-10.0±7.0 *	9.00±5 *** 2.00±5.0 3.00±4.0 -6.00±15
				Proprasylyte 2mg/kg (N=6)
2 5 10 30	-31.0±13 *-30.0±13 ***-33.0±10 ***-32.0±14 ***		-3.00±1.0 3.00±7.0 3.00±8.0 3.00±9.0

With before the administration relatively: ^*P＞0.05, ^*P＜0.05, ^{* *}P＜0.01

5. myocardium peptide element is to the influence of myocardial infarction

Body weight 20.9 ± 4.0kg adult healthy miniature pig, male, ear vein is injected 3% vetanarcol 30mg/kg anesthesia.Trachea cannula connects SC-3 type electric pulmotor pedestrian worker positive pressure respiration.Left side III intercostal is opened chest, exposes heart, separates anterior descending coronary (about 1/3 position of centroid point), wears 0 under it ^#Silk thread is in order to ligation; Myocardial surface is placed the fixed epicardial lead of multiple spot of 20 points under ligature, myocardial electrical signals is recorded in RM-6300 type eight through ZYS1-I type numerical control visceral pericardium scanner, AB-601G bioelectric amplifier and leads on the RTA-1200 type heat battle array registering instrument of physiograph, normal voltage 1mV=1mm, the visceral pericardium electrocardiogram(ECG of 20 points of self-timing mapping changes.Femoral arteriography connects the AP-641G blood pressure amplifier through TP-400T type pressure transducer and measures mean arterial pressure (MBP) to aorta abdominalis; The subcutaneous insertion needle electrode of four limbs through AC-601G ecg amplifier measurement standard II lead electrocardiogram (ECGII), and with ECG electric signal input AT-601G cardiotachometer, is measured heart rate (HR).Femoral venous catheter is used for administration and fluid infusion.

Experiment divides 4 groups, 25 of shared animals, the solvent control group is when venoclysis N.F,USP MANNITOL 120mg/kg, have 5 to survive 5 because of the chamber death of quivering, all the other respectively organize 5 every group, through femoral vein difference infusion cardiac muscle peptide element 5,10mg/kg, positive drug verapamil 0.25mg/kg, the administration volume is 2ml/kg, and infusion velocity is 2ml/min.Operation finishes and treats to trace the visceral pericardium electrocardiogram(ECG after every index is stablized, ligation descending anterior branch subsequently, record visceral pericardium electrocardiogram(ECG contrasts before as administration behind the 5min, intravenous infusion administration then, after the record administration 5,10,15,20,25,30,45,60,90,120, the visceral pericardium ECG ST section lift-off value of ECGII, MBP, HR and 20 points of 180min, and try to achieve summation (ST), with this index as the measurement degree of myocardial ischemia.The point that the rising of visceral pericardium ECG ST section is surpassed 2mV is decided to be the ischemic point, calculates total ischemic and counts (NST), with this index as the myocardial ischemia scope.3h sacrificed by exsanguination animal after the administration, immediately take out heart, cut ventricle, clean remained blood, the following ventricle in ligation position is pressed the thick crown section of 5mm, 1%TTC lucifuge dyeing 30min under the room temperature, again 5 double-edged ischemic regions of cardiac muscle and non-ischemic region are traced on transparent film, clip white infarct district film is weighed, and is heavy divided by 10 center of area chamber sarcoglia sheets, calculates the per-cent that infarct accounts for ventricular weight under the ligature.

Experiment grouping, dosage and administering mode

Group	Medicine	Infusion dosage (mg/kg)	Infusion velocity (ml/min)
				The solvent control group is tried the thing low dose group and is tried object height dosage group positive drug control group	N.F,USP MANNITOL cardiac muscle peptide element+N.F,USP MANNITOL cardiac muscle peptide element+N.F,USP MANNITOL verapamil	120 5+120 10+120 0.25	2 2 2 2

Table 23 venoclysis cardiac muscle peptide element is to the influence of pig myocardium infarction size

Medicine	Dosage (mg/kg)	Number of animals (n)	Infarction size (%)
				The plain verapamil of solvent control cardiac muscle peptide wish carnosine	- 5 10 0.25	5 5 5 5	19.4±3.02 11.8±3.13 * 10.2±3.2 ** 12.5±3.4 *

Annotate: compare with the solvent control group: ^*P＜0.05, ^*P＜0.01.

Evidence, cardiac muscle peptide element 5,10mg/kg can obviously reduce the Electrocardiographic ST of myocardial infarction Pigs Hearts adventitia, reduce NST, and dwindling myocardial infarct size, dead due to being quivered in irregular pulse, the chamber of the appearance of acute myocardial ischemia pig have certain therapeutic action, and blood pressure, heart rate are not had obvious influence.

Embodiment 3

Getting 1 kilogram of certified milk pig ventricular muscles cleans, chopping, add 1 kilogram of sterile purified water with the homogenate of 3000rpm/min rotating speed, homogenate is freezing 24 hours at-20 ℃, after thawing 3 times repeatedly, water proof is heated to 75 ℃, with model is sheet frame filter (available from the Guangzhou medicine instrument institute) filter cleaner of XAS03-172/8, select 10u middling speed filter paper for use, obtain coarse filtration liquid, (specification is F60 to coarse filtration liquid with hollow fiber column, available from Switzerland Zinpro Corp.) ultrafiltration, obtaining molecular weight is the smart filtrate of 12Kd, with ultra-filtration membrane (specification is 10Kd, Millipore company) ultrafiltration, molecular weight cut-off is the myocardium peptide cellulose solution 150ml of 9500Da, with the reverse osmosis concentration post concentrated product of Millipore company.

The solution process quality inspection of described myocardium peptide element is to meeting quality standard, filtration sterilization then, can, again through lyophilize, equipment used is a freeze drier, makes that shelf temperature reaches-20 ℃ in the kiln in 20 minutes, through 30 minutes, make products temperature reach-35 ℃ again; Keep temperature in the condenser being dropped to-50 ℃ in 2 hours, vacuumize again, when vacuum tightness reaches 100KPa, be communicated with kiln and condenser, stop the refrigeration of loft drier, when the vacuum tightness of loft drier begins to heat up during for 15Pa, heat-up rate is 3 ℃/min, is warming up to 15 ℃, is incubated 3 hours, continuation is warming up to 22 ℃ with 10 ℃/min speed, continues 5 hours; Continuation is warming up to 35 ℃ with 10 ℃/min speed, continues 2 hours; Continuation is warming up to 50 ℃ with 5 ℃/min speed, continues 1 hour.Enter temperature-fall period, make temperature reduce to 40 ℃ in the 20min, continue 10 hours, promptly obtain the plain dried frozen aquatic products of the qualified myocardium peptide of outward appearance, take out goods and seal.

Described myocardium peptide element by analysis, content of peptides is 85%, total free aminoacids is 8%, rna content 1%, thymus nucleic acid content 6%, molecular weight 9500 dalton.

Embodiment 4

Getting 1 kilogram of certified milk ox ventricular muscles cleans, chopping, add 1 kilogram of sterile purified water with the homogenate of 5000rpm/min rotating speed, homogenate is freezing 48 hours at-30 ℃, after thawing 4 times repeatedly, water proof is heated to 90 ℃, with model is sheet frame filter (available from the Guangzhou medicine instrument institute) filter cleaner of XAS03-172/8, select 8u middling speed filter paper for use, obtain coarse filtration liquid, (specification is F60 to coarse filtration liquid with hollow fiber column, available from Switzerland Zinpro Corp.) ultrafiltration, obtaining molecular weight is the smart filtrate of 12Kd, with ultra-filtration membrane (specification is 5Kd, Millipore company) ultrafiltration, molecular weight cut-off is the myocardium peptide cellulose solution 150ml of 5000Da, with the reverse osmosis concentration post concentrated product of Millipore company.

The solution process quality inspection of described myocardium peptide element is to meeting quality standard, filtration sterilization then, can, again through lyophilize, equipment used is a freeze drier, makes that shelf temperature reaches-18 ℃ in the kiln in 40 minutes, through 20 minutes, make products temperature reach-25 ℃ again; Keep temperature in the condenser being dropped to-40 ℃ in 1 hour, vacuumize again, when vacuum tightness reaches 95KPa, be communicated with kiln and condenser, stop the refrigeration of loft drier, when the vacuum tightness of loft drier begins to heat up during for 12Pa, heat-up rate is 2 ℃/min, is warming up to 10 ℃, is incubated 5 hours, continuation is warming up to 25 ℃ with 16 ℃/min speed, continues 3 hours; Continuation is warming up to 30 ℃ with 15 ℃/min speed, continues 1 hour; Continuation is warming up to 60 ℃ with 8 ℃/min speed, continues 2 hours.Enter temperature-fall period, make temperature reduce to 46 ℃ in the 30min, continue 8 hours, promptly obtain the plain dried frozen aquatic products of the qualified myocardium peptide of outward appearance, take out goods and seal.

Described myocardium peptide element by analysis, content of peptides is 78%, total free aminoacids is 15%, rna content 2%, thymus nucleic acid content 5%, molecular weight 5000 dalton.

Embodiment 5

Getting 1 kilogram of certified milk rabbit ventricular myocyte cleans, chopping, add 1 kilogram of sterile purified water with the homogenate of 1000rpm/min rotating speed, homogenate is freezing 72 hours at-10 ℃, after thawing 3 times repeatedly, water proof is heated to 85 ℃, with model is sheet frame filter (available from the Guangzhou medicine instrument institute) filter cleaner of XAS03-172/8, select 5u middling speed filter paper for use, obtain coarse filtration liquid, (specification is F60 to coarse filtration liquid with hollow fiber column, available from Switzerland Zinpro Corp.) ultrafiltration, obtaining molecular weight is the smart filtrate of 11Kd, with ultra-filtration membrane (specification is 3Kd, Millipore company) ultrafiltration, molecular weight cut-off is the myocardium peptide cellulose solution 150ml of 2000Da, with the reverse osmosis concentration post concentrated product of Millipore company.

The solution process quality inspection of described myocardium peptide element is to meeting quality standard, filtration sterilization then, can, again through lyophilize, equipment used is a freeze drier, makes that shelf temperature reaches-15 ℃ in the kiln in 10 minutes, through 25 minutes, make products temperature reach-30 ℃ again; Keep temperature in the condenser being dropped to-45 ℃ in 2.5 hours, vacuumize again, when vacuum tightness reaches 90KPa, be communicated with kiln and condenser, stop the refrigeration of loft drier, when the vacuum tightness of loft drier begins to heat up during for 10Pa, heat-up rate is 5 ℃/min, is warming up to 5 ℃, is incubated 6 hours, continuation is warming up to 15 ℃ with 8 ℃/min speed, continues 8 hours; Continuation is warming up to 32 ℃ with 7 ℃/min speed, continues 4 hours; Continuation is warming up to 55 ℃ with 4 ℃/min speed, continues 3 hours.Enter temperature-fall period, make temperature reduce to 50 ℃ in the 10min, continue 15 hours, promptly obtain the plain dried frozen aquatic products of the qualified myocardium peptide of outward appearance, take out goods and seal.

Described myocardium peptide element by analysis, content of peptides is 90%, total free aminoacids is 6%, rna content 1%, thymus nucleic acid content 3%, molecular weight 2000 dalton.

Embodiment 6

With embodiment 3, different is that molecular weight cut-off is the myocardium peptide cellulose solution of 4000Da, through quality inspection to meeting quality standard, filtration sterilization then, can, according to following prescription:

The plain 20mg of cardiac muscle peptide

N.F,USP MANNITOL 375mg

Gac 0.005mg

Water for injection adds to 5ml

Bottling places freeze drier, by making that shelf temperature reaches in the kiln in 30 minutes-20 ℃, through 40 minutes, make products temperature reach-35 ℃ again, keep temperature in the condenser being dropped to-50 ℃ in 3 hours, vacuumize again, when vacuum tightness reaches 95Kpa, be communicated with kiln and condenser, stop the refrigeration of loft drier, when the vacuum tightness of loft drier begins to heat up during for 15Pa, heat-up rate is 3 ℃/min, is warming up to 10 ℃, is incubated 4 hours.Continuation is warmed up to 20 ℃ with 12 ℃/min speed, continues 5.5 hours.Continuation is warmed up to 30 ℃ with 12 ℃/min speed, continues 1.5 hours.Continuation is warmed up to 60 ℃ with 6 ℃/min speed, continues 2 hours.Enter temperature-fall period, make temperature reduce to 48 ℃ in the 20min, continue 9 hours.Obtain the plain dried frozen aquatic products of the qualified myocardium peptide of outward appearance, take out goods and seal.

Lyophilize obtains myocardium peptide cellulose content 2.0mg/ml finished product.Described myocardium peptide element by analysis, content of peptides is 80%, total free aminoacids is 12%, rna content 2%, thymus nucleic acid content 6%, molecular weight 4000 dalton.

Embodiment 7

With embodiment 3, different is the ventricular muscles that raw material adopts the certified milk horse, molecular weight cut-off is the myocardium peptide cellulose solution of 8000Da, gained cardiac muscle peptide element by analysis, content of peptides is 84.5%, and total free aminoacids is 6%, rna content 2%, thymus nucleic acid content 7.5%, molecular weight 8000 dalton.

Embodiment 8

With embodiment 3, different is also to contain trehalose in the myocardium peptide cellulose solution, and the set of dispense ratio is: the plain 15mg/ml of myocardium peptide, trehalose 200mg/ml.

Embodiment 9

With embodiment 3, different is the ventricular muscles that raw material adopts health pig, also contains lactose in the myocardium peptide cellulose solution, and the set of dispense ratio is: the plain 18mg/ml of myocardium peptide, lactose 250mg/ml.

Embodiment 10

With embodiment 6, different is that molecular weight cut-off is the myocardium peptide cellulose solution of 1000Da, and wherein myocardium peptide cellulose solution component is:

The plain 16mg of cardiac muscle peptide

Sucrose 300mg

Gac 0.005mg

Water for injection adds to 5ml

Gained cardiac muscle peptide element by analysis, content of peptides is 82%, total free aminoacids is 12%, rna content 2%, thymus nucleic acid content 4%, molecular weight 1000 dalton.

Embodiment 11

The plain preliminarily stabilised test of cardiac muscle peptide

1. the result of the plain influence factor test of injection cardiac muscle peptide, accelerated test shows, is removing the rapid flavescence of appearance luster under outer packaging, humidity＞75% and the temperature＞37 ℃ condition.Moisture content increases, and vigor reduces.

2. room temperature keeps sample and investigates the result show that except that the outward appearance color and luster became little yellow, other project did not have change in the time of 480～540 days, stored under 4 ℃ of conditions, and each investigates the project no change.Show that injection cardiac muscle peptide element stored under humidity 45%～90% and room temperature condition 150 days at least, appearance character, content and vigor do not have change, under 4 ℃ of conditions, can store at least 480 days.

Claims (18)

1, a kind of preparation method of myocardium peptide element, it is characterized in that 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 times, be heated to 65～95 ℃ of filter cleaners, obtain coarse filtration liquid with the filtration of sheet frame filter, use hollow fiber column ultrafilter again, obtain smart filtrate, use the ultra-filtration membrane ultrafiltration, hold back the myocardium peptide cellulose 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.

2, the preparation method of myocardium peptide element according to claim 1 is characterized in that describedly not comprising that people's healthy Mammals comprises pig, ox, sheep, rabbit, horse.

3, the preparation method of myocardium peptide element according to claim 2 is characterized in that described Mammals is a young mammals, comprises sucking pig, cow, milk goat, newborn rabbit, newborn horse.

4, the preparation method of myocardium peptide element according to claim 3 is characterized in that described young mammals is a sucking pig.

5, the preparation method of myocardium peptide element according to claim 1, the add-on that it is characterized in that described sterile purified water are 0.5～4 times of mammiferous ventricular muscles; The rotating speed of described homogenate is 1000～5000rpm/min.

6, the preparation method of myocardium peptide element according to claim 1 is characterized in that described freezing for being lower than under-5 ℃ the temperature freezing 24～72 hours; Described type of heating is for adopting water proof heating or direct heating, and temperature is 70～90 ℃, and the time is for being no more than 2 hours.

7, the preparation method of myocardium peptide element according to claim 6 is characterized in that described freezing for descending freezing 36～48 hours at-20～-30 ℃; Described type of heating is for adopting the water proof heating, and temperature is 75～80 ℃, and the time is for being no more than 1 hour.

8, the preparation method of myocardium peptide element according to claim 1 is characterized in that described sheet frame filter selects for use less than 10 μ middling speed filter paper; With obtaining the smart filtrate of molecular weight behind the hollow fiber column ultrafilter less than 12kDa; Damming with the ultra-filtration membrane ultrafiltration obtains the smart filtrate of molecular weight less than 10kDa, concentrates with the reverse osmosis concentration post.

9, the preparation method of myocardium peptide element according to claim 8 is characterized in that described sheet frame filter is selected for use and is less than or equal to 5 μ middling speed filter paper.

10, the preparation method of myocardium peptide element according to claim 1 is characterized in that freezing dry process is: made the interior shelf temperature of kiln reach-15～-20 ℃, and again through 20～40 minutes, made products temperature reach-25～-35 ℃ in 5～40 minutes; Keep temperature in the condenser being dropped to-40～-50 ℃ in 1～3 hour, vacuumize again, when vacuum tightness reaches 90～100KPa, be communicated with kiln and condenser, stop the refrigeration of loft drier, begin to heat up when the vacuum tightness of loft drier is 10～15Pa, heat-up rate is 2～5 ℃/min, be warming up to 5～15 ℃, be incubated 3～6 hours; Continuation is warming up to 15～25 ℃ with 8～16 ℃/min speed, continues 3～8 hours; Continuation is warming up to 30～35 ℃ with 7～15 ℃/min speed, continues 1～4 hour; Continuation is warming up to 50～60 ℃ with 4～8 ℃/min speed, continues 1～3 hour; Enter temperature-fall period, make temperature reduce to 40-50 ℃ in 10～30min, continue 8-15 hour, obtain the plain dried frozen aquatic products of the qualified myocardium peptide of outward appearance.

11, the preparation method of myocardium peptide element according to claim 10 is characterized in that freezing dry process is: made the interior shelf temperature of kiln reach-18～-20 ℃, and again through 25～35 minutes, made products temperature reach-30～-35 ℃ in 20～30 minutes; Keep temperature in the condenser being dropped to-40～-50 ℃ in 1～3 hour, vacuumize again, when vacuum tightness reaches 90～100KPa, be communicated with kiln and condenser, stop the refrigeration of loft drier, begin to heat up when the vacuum tightness of loft drier is 10～15Pa, heat-up rate is that 3～4 ℃/min speed is warmed up to 8～12 ℃, is incubated 4～5 hours; Continuation is warmed up to 18～22 ℃ with 10～12 ℃/min speed, continues 4～6 hours; Continuation is warmed up to 33～35 ℃ with 9～12 ℃/min speed, continues 1.5～2 hours; Continuation is warmed up to 54～58 ℃ with 5～7 ℃/min speed, continues 1.5～2 hours; Enter temperature-fall period, make temperature reduce to 45～48 ℃ in 15～20min, continue 9～12 hours, obtain the plain dried frozen aquatic products of the qualified myocardium peptide of outward appearance.

12, the preparation method of myocardium peptide element according to claim 1 is characterized in that can adding vehicle when lyophilize, and its weight ratio consists of:

Cardiac muscle peptide element 15～20

Vehicle 100～375.

13, the preparation method of myocardium peptide element according to claim 12 is characterized in that can adding vehicle when lyophilize, and its weight ratio consists of:

Cardiac muscle peptide element 18～20

Vehicle 200～375.

According to the preparation method of claim 12 or 13 described myocardium peptide elements, it is characterized in that 14, described vehicle is N.F,USP MANNITOL, trehalose, lactose, sucrose or other freeze-drying auxiliary material.

15, the preparation method of myocardium peptide element according to claim 14 is characterized in that, described vehicle is a N.F,USP MANNITOL.

According to the preparation method of any one described myocardium peptide element of claim 2-11, it is characterized in that 16, the content of peptides of described myocardium peptide element is 75%～90%, total free aminoacids is 6%～15%, and rna content is less than 2%; Thymus nucleic acid content is less than 7.5%, and molecular weight is less than 10000 dalton.

17, the preparation method of myocardium peptide element according to claim 16 is characterized in that, the molecular weight of described myocardium peptide element is 1000～10000Da; Described myocardium peptide element shows 2-6 bar colored zone in isoelectric focusing electrophoresis, wherein pI be 10.92 be with painted darker; There is a stable maximum absorption band at described myocardium peptide element 190-210nm place in ultra-violet absorption spectrum; The plain vigor of described myocardium peptide is at least 2.2; Do not contain protein in the described myocardium peptide element; Described myocardium peptide element is analyzed through FPLC, and myocardium peptide element mainly contains 5 component peaks, and the percentage area adds up to 90%～95% relatively.

18, the preparation method of myocardium peptide element according to claim 17 is characterized in that, described myocardium peptide element has a maximum absorption band at ultra-violet absorption spectrum 200 ± 2nm place; The plain isoelectric focusing electrophoresis of described myocardium peptide shows 2 bands, wherein pI be 10.92 be with painted darker.

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