CN1337403A - Active monopeptide prepared from active blood polypeptide and its prepn. activity and sequence - Google Patents
Active monopeptide prepared from active blood polypeptide and its prepn. activity and sequence Download PDFInfo
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Abstract
The present invention discloses an active monopeptide separated from active blood polypeptide-calf blood deproteinized extract, its preparation process and its activity and sequence detection; it uses the created activity detemination method to screen activity of every separated component and analyze their bio-chemical property. Said invention adopts modern liquid-phase chromatography, including gel chromatography, C18 and C4 reversed phase chromatography and Silica and CN positive chromatography to separate and purity active component in the calf-blood deproteinized extract; and it utilizes high-performance liquid-phase chromatography, high-performance capillary electrophoresis and mass spectrometry to identify that it is an active peptide and determines its molecular weight. Said active peptide is mainly used for curing cerebrovascular disease.
Description
The invention discloses a kind of bioactive peptide and preparation method thereof, particularly the purposes of isolating active peptide and preparation method thereof, active detection and sequential analysis and this bioactive peptide from active blood polypeptide-calf blood protein-removed extraction.
Peptide is a class biologically active substance.It relates to a plurality of fields such as hormone nerve growth and reproduction, and its importance is the functional activity of each system, organ and cell in the control agent.The research of biologically active peptides is one of most active fields in the current biological study.In the 60 to 70's of twentieth century, hypophysis and more hypothalamic peptide hormone separation and purification obtain important breakthrough, and the separation and purification of middle nineteen seventies endogenous opiatepeptide is achieved success, and have promoted the development of neuropeptide research greatly.Have been found that numerous biologically active peptidess, as neuropeptide, braingut petide, atrial natriuretic peptide or the like, the adjusting of the various functional activities of wide participation organism causes the attention of each related discipline.In the research of biologically active peptides, the separation and purification work that content of crucial importance and requisite is exactly a peptide, but because the not only complex structure of peptide own, and residing environment contains various macromolecular mixture often, so the separation and purification of peptide is that the biology worker extremely pays close attention to the problem that studies for a long period of time always.
The general residing environment of biologically active peptides often various sized molecules mix body, the purpose of separation and purification is for further its composition of research, structure, biological activity, function and the mechanism of action etc., and further by the synthetic this peptide of the method for synthetic.Peptide content is very low, therefore so far none separately or the ready-made method of a cover can from the mixture of complexity, extract any polypeptide.
State in the journey in the separation and purification of biologically active peptides, critical step is to set up the biological activity determination method that all will use from start to finish, and this measuring method for activity is believable all the time, to guarantee to be purified into the west of coming that can provide signal in surveying the experiment of living.The basis of separation and purification peptide mainly is a difference of utilizing various characteristics between the molecule, as bulk of molecule and shape, soda acid character, solubleness, adsorption property with to the biology avidity of other molecule.The separation and purification pattern that now is used for purified peptide mainly contains three kinds: the one, and gel chromatography separates by the size of peptide molecule; The 2nd, ion-exchange chromatography separates by the character number of the charged gene that peptide molecule had; The 3rd, reverse-phase chromatography separates by the hydrophobic power of peptide molecule.From the extremely complicated mixture of composition, peptide is separated, usually can be utilized the different qualities (volume, charging property, hydrophobicity) of compound molecule to select for use the chromatographic process of several modes continuously to reach effective separation and purification.But always there is sample segment to lose inevitably in the sepn process in each step, should avoids separating step too much when very low as the concentration of peptide.Correct experimental design is to select for use minimum separating step to reach optimal separating efficiency.So for the rapid separable programming of multistep.Select the kind and the use order of pillar extremely important.Should select the suitable separation and purification pattern of a cover to obtain the pure product of high-purity high-activity for each peptide.For example in " application of high performance liquid chromatography in life science; scientific and technical literature press; 1998 ", disclose and utilized gel chromatography, ion-exchange chromatography and the reverse-phase chromatography difference on selectivity, adopt gel chromatography (Bio-Rad250), ion exchange column (SynChropakS300), reversed-phase column (SgnChropak RP-C18), from exempt from bone road flesh troponin, isolate needed peptide molecule fragment fully.
The kind of active blood polypeptide is a lot, generally be from the blood of animal, to separate to purify out, as calf blood protein-removed extraction (Deproteinized Hemoderivation Calfblood) is to be raw material with little ox blood, and molecular weight is less than 5000 daltonian small molecule bioactive preparations.
Calf blood protein-removed extraction (solcoseryl) nineteen fifty-five is at first developed by the plain high pharmaceutical factory of Switzerland (SOLCOBASLE A.G.), widespread use has been for many years in countries in the world, the Solcosery Eye Gel (Solcoseryl) that at present main listing product has the plain high pharmaceutical factory of Austrian Metrizamide pharmaceutical factory (HAFSLUND NYCOMEDAG) actovegin (Actovegin) and Switzerland to produce, and by the product of numerous manufacturers production of Japan and China.Its preparation has injection liquid, tablet, eye ointment, freezes cream etc., and injection liquid contains the 40-50mg calf blood protein-removed extraction for every milliliter.
The main pharmacological of calf blood protein-removed extraction is to promote picked-up and the utilization (do not rely on Regular Insulin) of cell to oxygen and glucose, this acts on functional defect, metabolism and is subjected to the situation (low blood oxygen, matrix deficiency) that presses down down and particularly outstanding during the energy requirement increase, make the anaerobic metabolism of glucose turn to aerobic metabolism, impelling energy matter to generate increases, prolong the cell rise time, promote histocyte metabolism and functional rehabilitation and tissue repair.As brain metabolism revivifier, be mainly used in treatment senile dementia (comprising Alzheimer disease, multi-infarct dementia etc.), acute and chronic cerebrovascular disease, the inaccessible disease of some illnesss of apoplexy, craniocerebral trauma, spinal cord, cerebral trauma, cerebral organic psychosis, peripheral blood vessel particularly, in addition, also be used for the treatment of various types of wounds, ulcer wound surface, burn, chemical burns, radiation injury etc., have good and clinical curative effect.
The composition of calf blood protein-removed extraction is very complicated, in its dry-matter, be mainly inorganics and small organic molecule, comprise the intermediate product of amino acid, carbohydrate, small molecular weight polypeptide, nucleosides, ketone acid and lipid metabolism etc., do not contain antigenicity substance, pyrogen and any propagable infectant.The data relevant with calf blood protein-removed extraction seen J Cell Physiol 1991 Jan, 146 (1): 681-5; Eksp Kiln Farmakol, 1999,62 (2): 61-3; Acta Physiol Hung 1996; 84 (1): 63-72; Neuropsychobiology 1998,37 (1): 28-35; Angiology 1992 Jan; 43 (1): 47-58; Arzneimittelforschung 1996 Mar, 47 (3): 269-72; EP824,375; US4,866,033.
Biochemical drug as a kind of determined curative effect, with the activeconstituents that plays a major role in the calf blood protein-removed extraction---a kind of bioactive peptide separation and purification is come out, to help its character, function, the mechanism of action are carried out further research and inquirement, and might carry out chemosynthesis according to separated structures.But since nineteen fifty-five, calf blood protein-removed extraction appeared on the market, its research has mostly been concentrated on its pharmacological action and clinical application aspect, do not see the therefrom report of separation and purification activeconstituents.
The object of the present invention is to provide a kind ofly by active blood polypeptide, particularly the bioactive peptide composition that goes out of calf blood protein-removed extraction separation and purification is further determined its activity, molecular weight, sequence and other physico-chemical property.
It is a kind of by active blood polypeptide that another object of the present invention is to provide, particularly the method for calf blood protein-removed extraction separation and purification bioactive peptide composition.
A further object of the present invention is to disclose the purposes of this bioactive peptide at aspects such as the treatment cerebrovascular and wounds.
In recent years, eukaryotic cell has been obtained very big progress from the extracellular to the research work of intracellular information transmission.People have grasped the basic structure of cell internal information transfer system, and these information transmission channels mainly are made up of a series of links such as functional protein, a series of protein kinase, target function albumen and multiple modulin of specific receptors, G albumen, release second messenger.In addition, extracellular information (as the matrix hormones) also can be by ionic channel or is directly imported in the cell, acts on corresponding system and causes cell response.By analysis to calf blood protein-removed extraction composition and pharmacological mechanism, estimate that its activeconstituents probably is bioactive peptide, possible mechanism is that bioactive peptide acts on cell surface receptor or other susceptor, cell passes to corresponding reactive system through a series of converting systems, thereby causes the energy metabolism of intracellular plastochondria to change.
Based on above-mentioned analysis, the present invention has at first set up Wa Shi respirometer manometry and has measured active, to brain metabolism revivifier---the calf blood protein-removed extraction raw material carries out screening active ingredients, determine to be suitable for isolating raw material, the present invention adopts the stratographic method, through gel chromatography, Clg and C4 reverse-phase chromatography, Silica and the separation and purification of CN normal-phase chromatography multistep liquid chromatography, each step isolate is followed the tracks of determination of activity with Wa Shi respirometer manometry, from calf blood protein-removed extraction injection, obtained a biological bioactive peptide, this bioactive peptide Toplink promotes picked-up and the utilization of cavy liver cell to oxygen, be the effective constituent in the deproteinized calf blood hydrolyzed solution injection liquid, further adopt HPCE to identify purity, HPGC working sample molecular weight, and measured its UV spectrum and mass spectrum.
At first adopt Wa Shi respirometer manometry to measure the biological activity of four kinds of calf blood protein-removed extractions such as " solcoseryl injection liquids " of " short cell generates element " of " the Socolseryl injection liquid " of " the Actovegin injection liquid " of Austrian Hafslund Nycomed AG product, Switzerland Solco Basle product, Jinzhou Medical College's product, Shanghai Li Bao Biology Pharmacy Co., Ltd product among the present invention.By determination of activity, screening the highest wherein active sample separates purification as the starting material that are used for separation and purification respectively through gel chromatography, C18 and C4 reverse-phase chromatography, Silica and CN normal-phase chromatography; After above-mentioned each step separation and purification, each the fraction sample that obtains is after lyophilize, adjusting concentration, carry out active tracking and measuring determining active ingredient, the component that the activity that obtains is the highest is carried out next step again and is separated, and goes out activeconstituents in the calf blood protein-removed extraction to separation and purification; Identify that with high performance liquid chromatography, HPCE it is a bioactive peptide, its molecular weight has been determined in mass spectroscopy, and the Edman edman degradation Edman has been determined its aminoacid sequence.The detailed process of separation alcoholization as shown in figure 27.
Wa Shi respirometer manometry is a kind of in the closed system of constant temperature constant volume, utilizes the technology of the gaseous interchange of trace in variation of gas pressure quantitative assay biomaterial or some chemical reaction.In the present invention, when utilizing the cellular respiration of cavy liver homogenate, consume O
2, emit CO simultaneously
2, and CO
2The KOH that is added in the reaction flask absorbs, and therefore manometric pressure change is by O in the reaction flask
2Minimizing produced, can measure O by the variable quantity of pressure
2Absorbed dose, calculate the activity of sample or separated portion thus.The determination of activity of four kinds of calf blood protein-removed extractions, Actovegin injection liquid activity in four kinds of samples is the highest, the starting material of Actovegin injection liquid as separation and purification are adopted in decision, in addition, selecting the Actovegin injection liquid is that other auxiliary material in this sample helps separation and purification as the further Another reason of separate raw materials.
For pattern and the condition of selecting separation and purification better, the present invention has measured the molecular weight as the raw-material Actovegin injection liquid of separation and purification.Gel chromatography and electrophoretic method are laboratory molecular weight determinations commonly used, but electrophoretic method generally can only the above compound of determining molecular weight 5000 dalton, so this experiment employing high performance liquid phase gel chromatography molecular weight analyte; Because of the Actovegin injection liquid is the small molecules mixture, be that molecular weight 100～7000 daltonian Superdex Peptide HR 10/300 gel chromatographic columnses are measured its molecular weight so adopt separating ranges.With the reference material of known molecular amount, on same gel chromatographic columns, carry out the glue chromatography with the same terms.Owing to add the molecular weight of material difference of gel chromatographic columns, so elution volume is also inequality.Obtain the Kav value according to elution volume, and have linear relationship (Kav=-blgMW+c) between Kav value and the molecular weight, therefore, can draw a typical curve.Then, under similarity condition, can obtain its Kav value, be worth on typical curve with this and just can obtain its molecular weight by the elution volume of unknown molecular quantity of material.With high performance liquid phase gel chromatography molecular weight, has the advantage that finding speed is fast, amount of samples is few.The molecular weight 95% that obtains the Actovegin injection liquid is less than 1500 dalton.
For the further selection for separation and purification pattern and condition provides information, utilize efficient capillary zone electrophoresis (HPCZE) analysis that the Actovegin injection liquid has been carried out polynary qualitative analysis among the present invention.HPCE (HPCE) analysis is the higher analysis means of present resolving power.Capillary zone electrophoresis (CZE) is meant in the background electrode buffer of solute in kapillary with the friction-motion speed migration and forms an independently electrophoretic of solute band, and it separates basis is each solute difference between net charge and mass ratio in electrode buffer.The CZE pattern is a kind of operator scheme most widely used among the HPCE.The selection of electrode buffer directly has influence on the migration of solute and last separating, and its pH value at least must be than the high or low PH unit of the iso-electric point of analyte.In general, as long as conditions permit just adopts acid electrode buffer as far as possible, the iso-electric point of in fact all peptides is all greater than PH4.
From isolating angle, each step separation of polypeptide will be found optimal separation method and separation condition as far as possible, improves the treatment capacity and the rate of recovery of sample.But biologically active substance for unknown its biochemical property of separation and purification from mixture, and when monitoring separated portion with the method for determination of activity, the more important thing is and to notice that activeconstituents can not be dispersed in different the collection in the part, because it is activeconstituents may be made of two or more materials, time saving and energy saving when separating at last than beginning; Therefore the present invention adopts following separation purification method:
1. mesolow liquid chromatography (FPGC) is separated calf blood protein-removed extraction:
In biochemistry separates, often adopt the first step of ion exchange chromatography chromatography as separation and purification, because it is very convenient and efficient is higher to use large vol to prepare the type ion exchange column in the purifying of preparation property, but for the present invention, because salt concn too high (silver nitrate titration method is measured chlorine ion concentration 6mg/ml) in the Actovegin injection liquid, and the too little methods such as gel-filtration or dialysis of can't using of molecular weight carry out desalting treatment, so this experiment adopts the HiLoad 26/60 Superdex post of mesolow liquid chromatograph (FPGC) and preparation type to carry out the first step of gel chromatography separation as the separation and purification of Actovegin injection liquid.Gel chromatography is that the difference of and shape big or small according to compound realizes isolating a kind of technology.The separation mechanism of gel chromatography can illustrate that promptly small molecules and solvent can enter in the hole of fixed phase stuffing fully with solid eliminating mechanism, is called infiltration fully, its partition ratio K=1; Macromole can not enter fully, is called fully to get rid of its partition ratio K=0; Molecule for middle size then belongs to selective permeation, it can enter in some hole of filler, and can not enter other fillers than in the aperture, so its partition ratio K is between 0～1, this part is the effective scope of gel chromatography separation, in this scope, if the indicating correct of molecular weight and do not consider the influence of shape of molecule then will be linear relationship between the logarithm of molecular weight and the elution volume.
In gel chromatography, the very important point is to select suitable column packing.The selection of column packing can from following some consider: 1. should select suitable column packing according to the molecular weight ranges of separated material.The compounds of different sizes when their elution volume during near the intermediate value of filler separating ranges, have best separation.2. select column packing with appropriate bore volume distributed median.The big more peak capacity of pore volume is big more, separate also high more, but pore volume when too big filler not withstand voltage, as dextrane gel, sepharose, withstand voltage is less than 0.3MPa, is unwell to HPLC and goes up and use.3. the column packing particle is thin more, and size is even more, and resolving power is high more, but back-pressure is also higher, and is also high more to the requirement of filled column and system.4. physical stability and chemical stability.Good physical stability just can be born high flow rate, keeps column volume and Stability Analysis of Structures simultaneously, improves stratographic repeatability; High chemical stability promptly refers to adapt to the PH concentration and the organic solvent of broad, and it helps developing separation condition and cleaning on the throne.According to the character of Actovegin injection liquid, among the present invention, at first selected mesolow liquid phase systems and HiLoad 1.6/60 Superdex post that the Actovegin injection liquid is carried out the first step gel chromatography.HiLoad 1.6/60 Superdex post, column volume 120ml, granular size 22～44um, separating ranges＜10000 dalton, withstand voltage 0.3Mpa, medium are dextran covalent cross-linking agarose, combine the highly selective of dextran and the high physical stability of agarose, can modification under high flow rate, batch processing repeatability is fine.Experimental result shows that the FPGC method is separated six kinds of components that obtain, and wherein the relative reactivity of F5 group is much higher than other component, illustrates that the F5 component is the component that activeconstituents mainly exists.Therefore among the present invention, proceed to separate with the F5 group that isolated activity is the highest.
2. efficient reversed-phase liquid chromatography (HPPC) separates the F5 component:
HPRPC is that separate the small molecules biochemical substances the most frequently used also is one of effective means.Make moving phase and desolvate and characteristics such as aftertreatment is more convenient because it has resolution height, good reproducibility, employing organic solvent than easy from sample, the removing of brine system.Silica gel bonded phase filling is the most frequently used stationary phase of HPRPC.The filler physical strength of silica matrix is good, and the proof pressure height is applicable at HPLC.Silica gel bonded phase filling commonly used in HPRPC has 18 alkyl silica gel (C18 or ODS), octyl group silica gel (C8), benzyl silica gel (C4) etc.Second step of the present invention selects the C18 silicagel column to separate the highest component of activity that the first step obtains in separating.Fixedly the time, the bigger chromatogram that has that the bonding chain is long keeps in condition, but when the strong carbon number that closes chain surpasses 7, the carbon number increase, retention time does not change.HPRPC adopts the gradient elution mode to come sample separation more, and moving phase is made up of the organic solvent of water damping fluid and different concns usually.Acetonitrile is the most frequently used in the organic solvent, because it does not have uv-absorbing between 210～80nm, easily removes and when the isolating biologically active material, higher biological activity and mass recovery is arranged.Normal adding trifluoroacetic acid or hyptafluorobutyric acid plasma are to reagent in the moving phase of HPRPC, and the first increases compound and bonding hydrophobic interaction mutually, regulate to keep; Its two pH value that makes moving phase prevents silanol base residual in stationary phase ionization and cause second adsorption under alkaline condition between 2.5～5.0.
Second step of the present invention is separated the employing liquid chromatograph, Zorbax 300SB-C18 post, experimental result shows that HPRPC separates the relative reactivity in each component that obtains, wherein the relative reactivity of I component is 75.3%, be higher than other component, illustrate that the I component is the component that activeconstituents mainly exists, therefore select the I component to carry out the separation in the 3rd step.
3.I the efficient reversed-phase liquid chromatography (HPRPC) of component separates
Adopt and identical liquid chromatograph of second step, Vydac 300SB-C4 post carries out the further separation of I component, experimental result shows separates in 6 components that obtain, Y4 component relative reactivity 69.2%, be much higher than other component, illustrate that the Y4 component is the component that activeconstituents mainly exists, select the Y4 component to carry out for the 4th step and separate.
4.Y4 the efficient positive liquid chromatography (HPNPC) of component is separated
HPNPC is with the hydrophilic filler phase that fixes, and as the polar stationary phase of bonded hydroxy on silica gel, amino and cyano group, makes the liquid chromatography of moving phase with hydrophobic solvent or mixture.The separation of HPNPC be decided by solute and stationary phase and between Intermolecular Forces, wherein hydrogen bond force and electrostatic force play an important role.Among the present invention, with high performance liquid chromatograph, Zorbax 300SB-CN post, gradient elution, obtain six component Y, experimental result shows that in isolated six components, Z3 component relative reactivity 85.4% is higher than other component far away, illustrate that the Z3 component is the component that activeconstituents mainly exists, can be used for carrying out the separation and purification in the 5th step.
5.Z3 efficient positive liquid chromatography (HPNPC) purifying of component
Use high performance liquid chromatograph, the gradient elution separation Z3 component of Zorbax RX-SIL post, obtain four groups of isolate Z, experimental result shows in the component of separating, W3 component relative reactivity is 50.7%, activity is higher than other component, illustrates that the W3 component is the component that activeconstituents mainly exists, and carries out next step purifying with W3.
6.W3 efficient positive liquid chromatography (HPNPC) purifying of component
Use high performance liquid chromatograph, Zorbax RX-SIL post separates, and obtains three components, and wherein the relative reactivity of P2 component is 49.6%, is higher than other component, and the P2 component is that activeconstituents mainly exists part.
From from the raw material calf blood protein-removed extraction, each goes on foot isolating separate colors spectrogram as can be seen, carries out along with isolating, and the purity of the component that obtains improves gradually; Spectrogram is by a plurality of eclipsed peak, and the peak after separating in the color atlas draws back gradually, obtains the several separate peak, to the final step separation, in the spectrogram that obtains, is a main peak substantially, only contains small amount of impurities; Owing to obtain the activity of sample simultaneously with the separation and purification of Wa Shi respirometer manometry tracking and measuring, can think to have obtained a kind of bioactive peptide in the calf blood protein-removed extraction.
Aforesaid method of the present invention also is applicable to isolating active list peptide from the active blood polypeptide of other animal.
The present invention adopts efficient capillary zone electrophoresis (HPCZE) to identify that isolated bioactive peptide P2 component purity is greater than 95%; High performance liquid phase gel chromatography (HPGC) determining molecular weight is that 803 roads pause; Amino acid composition analysis is an important component part of measuring peptide sequence, it not only provides prerequisite for formulating the order-checking scheme, and be that accuracy and the efficient that checks order plays effect, hydrochloric acid hydrolysis is the most widely used a kind of method of present protein hydrolysate and peptide, hydrochloric acid hydrolysis all destroys tryptophane, Threonine and Serine destroy 5～10%, the ruined reason of tryptophane is oxidation, if the phenol of adding 1%, generally can reach 70% the rate of recovery, the present invention adopts hydrochloric acid hydrolysis, column front derivation method assay P2 component amino acid to form.
The present invention has measured the ultra-violet absorption spectrum of bioactive peptide P2 component at 190～300nm.Polyenoid system in the polypeptide can absorb the UV-light in the 190-300nm wavelength region.There is different absorption peaks in different functional groups, therefore, the whole absorption spectrum of examination peptide can disclose about the amino acid of peptide form and the quantitative aspect of peptide for information about.Form if know the amino acid of peptide, then perhaps might estimate its purity by checking spectrum.In the ultraviolet wavelength scope of 190-220um, conjugated double bond such as peptide bond have the absorption of strong row, and other biological active material such as protein and nucleic acid also show extremely strong absorption, are more suitable with the active matter of the UV-light detection of biological in this wavelength region therefore.But this is not a feature, and other compound such as trifluoroacetic acid also have the absorption of class at 190-220nm.
The present invention measures through the liquid phase second order ms in addition, has determined molecular weight and tile structure information; And measure its sequence through automatic Protein Sequencer with the Edman edman degradation Edman.Above-mentioned analytical test proves that further isolated is a bioactive peptide.
Bioactive peptide of the present invention directly acts on cell for pavilion, promotes picked-up and the utilization (do not rely on Regular Insulin) of cell to glucose, increases the uptake rate and the utilization ratio of cell oxygen, thereby the generation of ATP is increased, and the cell energy amount is improved.This is a kind of effect that does not rely on organ.Cause the improvement of cellular metabolism by this effect, thereby improve cellular metabolism and functional status, this acts under the hypoxic-ischemic state particularly remarkable.Be subjected to the situation (low blood oxygen, matrix deficiency) that presses down down and energy requirement when increasing in functional defect, metabolism, promote the cellular energy metabolism, improve cell function, make the blood supply increase at cell levels.In addition, it and various human growth factor have synergistic effect, and tissue is had proliferation function.Be mainly used in treatment senile dementia (comprising Alzheimer disease, multi-infarct dementia etc.), acute and chronic cerebrovascular disease, the inaccessible disease of some illnesss of apoplexy, craniocerebral trauma, spinal cord, cerebral trauma, cerebral organic psychosis, peripheral blood vessel particularly, in addition, also be used for the treatment of various types of wounds, ulcer wound surface, burn, chemical burns, radiation injury etc.
The present invention's activity determination method---Wa Shi respirometer manometry of setting up, to brain metabolism revivifier---the calf blood protein-removed extraction raw material carries out screening active ingredients, determined to be suitable for separating the raw material of purification, use the modern liquid chromatography method, through gel chromatography, C18 and C4 reverse-phase chromatography, Silica and CN normal-phase chromatography, therefrom separation and purification goes out an activeconstituents, active polypeptide, this polypeptide proves through determination of activity, can promote of picked-up and the utilization of cavy liver cell to oxygen, be the effective constituent in the deproteinized calf blood hydrolyzed solution injection liquid, the present invention further uses high performance liquid chromatography, HPCE identifies that it is a bioactive peptide, its molecular weight has been determined in mass spectroscopy, the Edman edman degradation Edman has been determined its aminoacid sequence, methodological science of the present invention, reliably, isolated bioactive peptide is for further research brain metabolism revivifier---the function of calf blood protein-removed extraction, the mechanism of effect and treatment various diseases has great importance; Simultaneously, for adopting synthetic this bioactive peptide of biology and chemical process to lay a good foundation.The bioactive peptide that separation and purification goes out behind the aminoacid sequence, can carry out synthetic after measured, and if can be applied to clinically will have huge using value.
Describe the present invention in detail below in conjunction with the drawings and specific embodiments, described embodiment is used to describe the present invention, rather than restriction the present invention.
Description of drawings:
Fig. 1 is the HPGC collection of illustrative plates of molecular weight standard mixing solutions of the present invention;
Fig. 2 is a molecular weight standard curve of the present invention;
Fig. 3 is the HPGC collection of illustrative plates of Actovegin injection liquid of the present invention;
Fig. 4 analyzes collection of illustrative plates for the HPCZE of Actovegin injection liquid of the present invention;
Fig. 5 is the FPGC separating spectrum of Actovegin injection liquid of the present invention;
Fig. 6 respectively collects ratio of component than collection of illustrative plates for Actovegin injection liquid FPGC of the present invention separates;
Fig. 7 is the structure iron of Superdex of the present invention;
Fig. 8 is the HPRPC separating spectrum of F5 component of the present invention;
Fig. 9 is the isolating component of respectively collecting of F5 component HPRPC of the present invention;
Figure 10 respectively collects ratio of component for F5 FPGC of the present invention is isolating;
Figure 11 is the HPRPC separate colors spectrogram of I component of the present invention;
Figure 12 respectively collects part for I component of the present invention after HPRPC separates;
Figure 13 is each separate part relative volume, peak area and the active comparison of I component of the present invention;
Figure 14 is the HPNPC separate colors spectrogram of Y4 component of the present invention;
Figure 15 respectively collects part for Y4 component of the present invention after HPNPC separates;
Figure 16 is each separate part relative volume, peak area and the active comparison of Y4 component of the present invention;
Figure 17 is the HPNPC separate colors spectrogram of Z3 component of the present invention;
Figure 18 respectively collects part for Z3 component of the present invention after HPNPC separates;
Figure 19 is each separate part relative volume, peak area and the active comparison of Z3 component of the present invention;
Figure 20 is the purifying color atlas of W3 component of the present invention;
Figure 21 identifies collection of illustrative plates (in the electrode buffer of pH value 2.5) for P2 component HPCZE purity of the present invention;
Figure 22 identifies collection of illustrative plates (in the electrode buffer of pH value 8.0) for P2 component HPCZE purity of the present invention;
Figure 23 is a HPGC working sample P2 component molecular weight collection of illustrative plates of the present invention;
Figure 24 is a PTC-amino acid reference colour collection of illustrative plates of the present invention;
Figure 25 is the ultra-violet absorption spectrum of P2 component of the present invention;
Figure 26 is the mass spectrum of P2 component of the present invention;
Figure 27 is the schema by calf blood protein-removed extraction isolating active list peptide of the present invention;
Figure 28 is the isolating schema of calf blood protein-removed extraction.
Present embodiment relates to the activity of measuring the separation and purification sample with Wa Shi respirometer manometry.Specific as follows:
Material and instrument: Wa Shi respirometer: Warburg system 12062 types, German Braun-Melsungen produces; The no side arm reaction flask of marking; The high-speed homogenization machine; Animal is the heavy cleaning level male guinea pig of 250 ± 10g; Four kinds of calf blood protein-removed extractions are: the lot number that Austrian Hafslund Nycomed AG produces is 152842 Actovegin injection liquid; The lot number that Switzerland Solco Basle produces is 414409 Socolseryl injection liquid; The lot number that Jinzhou Medical College produces is 981216 short cell generation element; The lot number that Shanghai Li Bao Biology Pharmacy Co., Ltd produces is 990102 solcoseryl injection liquid.Reagent is: 10%KOH solution, 0.2mol/ml PBS and Brodie manometric liquid, the compound method of calf blood protein-removed extraction is: take by weighing Glycocholate sodium 5g, NaCI 23g dissolves the back respectively and merges, and adds Evans Blue 100mg, be settled to 500ml, it is anticorrosion to add several vanilla phenol spirituous solutions.
Experimental technique: the cavy fasting is after 24 hours, and neck hits puts to death, and takes out liver rapidly.Take by weighing hepatic tissue 4g, add 24ml 0.2mol/ml PBS, 6000rpm homogenate 30 seconds, the multilayer filtered through gauze obtains liver homogenate liquid, cleans to reach in advance and presses table 1 adding sample and reagent in the exsiccant reaction flask; Behind the reaction flask and corresponding side pressure pipe coupling that install, reaction flask is immersed in 37 ± 1 ℃ of waters bath with thermostatic control, start vibrator (100 times/minute) vibration 20 minutes, carry out the temperature and pressure compensation; Measuring cell right side post piezometric fluid level is transferred to the 150mm place, record left side manometric liquid reading (A
1), close the three-dimensional piston, start vibrator; After 15 minutes, close vibrator, utilize knob to regulate measuring cell right side post manometric liquid liquid level, record left side manometric liquid liquid level reading (E to the 150mm place
1); 4,5 processes twice of repetition, reading is recorded as A respectively
2, E
2, A
3, E
3
The relative reactivity method of calculation are as follows:
The calculating of reaction flask constant K value:
Wherein: V
1Be reaction flask volume (μ l)
1V
2Be piezometric tube volume (μ l);
*0.0239 O when being 37 ℃
2Solubleness.
Oxygen-consumption Δ O
2The calculating of (μ l):
ΔO
2＝[(A
l－E
1)＋(A
2－E
2)＋(A
3－E
3)]×K
The calculating of relative reactivity (%):
The relative reactivity measurement result of four kinds of samples being measured by Wa Shi respirometer manometry sees Table 1--4.By table 1--4 as can be seen, Actovegin injection liquid activity in four kinds of samples is the highest, and the starting material of Actovegin injection liquid as further separation and purification are adopted in decision, and in addition, other auxiliary material helps separation and purification in the Actovegin injection liquid.
The short cell of table 1. generates plain determination of activity result
Average relative reactivity is 99.04%
Table 2 solcoseryl injection liquid determination of activity result
Average relative reactivity is 153.697%
Table 3.Actovegin injection liquid determination of activity result
Average relative reactivity is 234.32%
Table 4.Socolseryl injection liquid determination of activity result
Average relative reactivity is 175.27%
Breathe in the manometry at Wa Shi, the problem that note has: when getting the cavy hepatic tissue, the position size is suitable, especially courage can not be broken; Before the homogenate hepatic tissue is cut into the even and little fritter of trying one's best of size, guarantees the homogeneous of homogenate; Begin this section period as far as possible rapidly and keep each time determination of activity time unanimity etc. from getting hepatic tissue to pressure measurement, this auspicious ability reduces error between each sample determination as far as possible.For guaranteeing determination of activity accuracy and tolerance range, Wa Shi breathes that the manometry experiment is each to be detected same sample and three parallel samples must be set and establish three parallel samples of Actovegin injection liquid reference substance, and detected result meets the following conditions: 1. barren oxygen-consumption Δ O2 〉=20 μ l; 2. the relative reactivity of reference substance 〉=150%, otherwise invalid.In addition, though each determination of activity expends more sample, in order to guarantee the exactness of measurement result, the result is repeated after the calibrating really active ingredient.
Present embodiment relates to high performance liquid phase gel chromatography (HPGC) measures the further raw-material Actovegin injection liquid molecular weight of separation and purification of conduct, specific as follows:
Material and instrument: high performance liquid chromatograph: Beckman 125NM pump, 126NM detector, workstation; SuperdexPeptideHR 10/300 post: 10mm * 30cm, column volume 24ml, column packing granular size 22～44um, separating ranges 100～7000 dalton, withstand voltage 1.5MPa, Pharmacia product; Moving phase: the aqueous solution of 0.05mol/L sodium phosphate+0.1mol/L sodium-chlor, PH7.0.
Standard specimen is: relative molecular weight 13700 daltonian two ribonuclease As, Pharmacia product; Relative molecular weight 5180 daltonian Regular Insulin, the Fisherbrand product; Relative molecular weight 1508 daltonian Somatostatins (14 peptide); Relative molecular weight 560 daltonian six peptides, the Biomol product; Sample is that the lot number of Austrian HafslundNycomed AG is 152842 Actovegin injection liquid.
Method: take by weighing an amount of ribonuclease A, Regular Insulin, 14 peptides and six peptides respectively and be mixed with the molecular weight standard mixing solutions; Chromatographic condition: flow velocity: lml/min, column temperature: room temperature, detect wavelength: 214nm, sample size: 20 μ l.
Experimental result is seen Fig. 1-3, and wherein Fig. 1 is the HPGC collection of illustrative plates of nuclease A, Regular Insulin, 14 peptides, six peptide molecular weight standard mixing solutionss, and Fig. 2 is the molecular weight standard curve, and the retention time of each standard specimen and logarithm molecular weight are:
Rt(min) lgMW
Nuclease A 13.93 4.1367
Regular Insulin 14.93 3.7634
14 peptides 16.87 3.1821
Six peptides 18.6 2.7482
The result shows under this gel chromatography condition, the molecular weight logarithm of molecular weight standard material and retention time linear substantially (R=0.9972), in conjunction with the HPGC collection of illustrative plates of Actovegin injection liquid, Fig. 3 obtains the molecular weight 95% of Actovegin injection liquid less than 1500 dalton.
Present embodiment relates to the efficient capillary zone electrophoresis (HPCZE) of Actovegin injection liquid and analyzes.
Material and instrument: capillary electrophoresis apparatus: Beckman P/ACE 5000, fixed wave length UV-detector, workstation; Vitreosil capillary column: Beckman FS 75um * 57cm (useful length is 50cm); Sample Actovegin injection liquid, lot number is with embodiment 1,2.
Method: preparation electrode buffer: get 1.7m185% phosphoric acid, add the 10ml acetonitrile, add water to 250ml, 1mol/l NaOH transfers pH value to 2.5, and the 0.45um membrane filtration is used preceding ultrasonication.Kapillary cleaned 5 minutes with 1mol/1NaOH before use successively, and ultrapure water cleaned 5 minutes, cleaned 2 minutes with 0.1mol/1NaOH before the sample introduction, and ultrapure water cleaned 1 minute, and electrode buffer cleaned 2 minutes.Adopt the high pressure input mode, sample injection time is 3 seconds, and the capillary sample inlet end is anodal, and the detecting end is a negative pole, and separation voltage is 20kv.The detection wavelength is 214nm, and the capillary column separation temperature is 25 ℃.
Experimental result is seen Fig. 4, utilizes HPCZE to carry out polynary qualitative analysis in the complex sample, and purpose is that the selection for separation and purification pattern and condition provides information.
Present embodiment relates to mesolow liquid phase gel chromatography (FPGC) and separates the Actovegin injection liquid.
Material and instrument: mesolow liquid chromatograph: AKTA Purifier 100, multi-wavelength UV-detector, PH/ electrical conductivity detector, automatic sampler, workstation; HiLoad 16/60 Superdex post: 16mm * 60cm, the Pharmacia product; Sample Actovegin injection liquid is with embodiment 1-3, vacuum freeze drier.
Method: at system stability, post is saturated, sample introduction after the balance, chromatographic condition is: moving phase: the aqueous solution of 10% acetonitrile+0.1%TFA, flow velocity is 1.2ml/min, column temperature is a room temperature, the detection wavelength is 214nm, detection is led in electricity consumption, autopipette sample introduction, sample size 1.5ml, valve is collected, by volume Fractional Collections F3, F4, F5, F6, each component of F7, F8, each component is after low pressure is taken out organic solvent, after the lyophilize, each component is after an amount of dilution, and Wa Shi respirometer manometry is measured activity respectively.Fig. 6 is the structure of Superdex post.
The result sees Fig. 5, Fig. 7 respectively, the result shows that the relative reactivity of F3, F4, FS, F6, F7, F8 component is respectively 9.1%, 5.3%, 97.5%, 0.6% ,-3.6% ,-2.4%, the relative reactivity of F5 component is much higher than other component, illustrates that the F5 component is the component that activeconstituents mainly exists.Therefore carry out next step separation with the F5 component.
The efficient reversed-phase liquid chromatography (HPRPC) that present embodiment relates to the F5 component separates:
Material and instrument: high performance liquid chromatograph: Shimadzu LC-10ATvp pump, DGU-12A de-aerator, CTO-10Asvp column oven, SPD-M10Avp detector, FRC-10A collector, SCL-10Avp controller, workstation; Zorbax 300SB-C18 post: 9.4mm * 25cm, DuPont company product.The aqueous solution of mobile phase A liquid: 0.1%TFA, pH value 2.5,0.45 μ m membrane filtrations are used preceding ultrasonic degas; Mobile phase B liquid: the aqueous solution of 0.1%TFA+80% acetonitrile, 0.45 μ m membrane filtration is used preceding ultrasonic degas.F5 component: after the vacuum lyophilization, be diluted to about 50mg/ml vacuum freeze drier with mobile phase A liquid.
Method: chromatographic condition is: detect wavelength 214nm, 37 ℃ of column temperatures, sampling volume 500 μ l, gradient elution, by each component of time Fractional Collections, order is compiled and is A, B, C, D, E, F, G, H, I, J, K, L, M, N, 0, P, Q, R, S, T, and each component is taken out the organic solvent postlyophilization through low pressure, each component after the lyophilize is after an amount of dilution, and Wa Shi respirometer manometry is measured activity respectively.Gradient is as follows:
Time (min) flow velocity (ml/min) B%
0.00 2.00 0
10.00 2.00 10
15.00 2.00 20
53.00 2.00 100
40.00 2.00 100
50?00 2.00 0
The result sees Fig. 8, Fig. 9, figure l0 respectively.The result shows that the relative reactivity of A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T component is respectively 0 9%, 10.2% ,-7.2%, 6.9%, 0.2%, 1.3%, 9.4%, 1.8%, 75.3%, 69% ,-23.0%, 2.5%, 26.5% ,-22.9%, 0.3%, 10.4%, 4.7%, 8.4%, 4.5% ,-9.3%.I component relative reactivity is much higher than other component, illustrates that the I component is the component that activeconstituents mainly exists, and therefore selects for use the I component further to separate purification.
The efficient reversed-phase liquid chromatography (HPRPC) that present embodiment relates to the I component that obtains among the embodiment 5 separates:
Material and instrument: adopt the high performance liquid chromatograph of embodiment 5, Vydac 300SB-C4 post: 6mm * 25cm, Vydac product.Mobile phase A liquid: the aqueous solution of 0.1%TFA+20% acetonitrile (pH value 2.80); The acetonitrile solution of Mobile phase B liquid: 0.1%TFA, moving phase are all used 0.45 μ m membrane filtration, use preceding ultrasonic degas; Sample: after the I component vacuum lyophilization, be diluted to about 3.5mg/ml with mobile phase A liquid.
Method: chromatographic condition is: detect wavelength 214nm, 37 ℃ of column temperatures, sampling volume 50 μ l, gradient elution, by each component of time Fractional Collections, order is compiled and is Y1, Y2, Y3, Y4, Y5, Y6 component, each component is after low pressure is taken out organic solvent, lyophilize, dried each component is measured activity respectively with Wa Shi respirometer manometry after an amount of dilution.Gradient:
Time (min) flow velocity (ml/min) B%
0.00 1.00 0
5.00 1.00 0
15.00 1.00 55
25.00 1.00 65
30.00 1.00 65
35.00 1.00 100
40.00 1.00 100
The result sees Figure 11, Figure 12, Figure 13 respectively.The relative reactivity that shows Y1, Y2, Y3, Y4, Y5, Y6 component is respectively-14.5%, 2.7%, 64%, 69.2%, 22.1%, 1.8%.Y4 component relative reactivity is higher than other component, illustrates that the Y4 component is the component that activeconstituents mainly exists, and therefore selects for use the Y4 component further to separate purification.
Present embodiment relates to the efficient positive liquid chromatography (HPNPC) of Y4 component and separates:
Material and instrument: high performance liquid chromatograph is the same, Zorbax 300SB-CN post: 4.6mm * 25cm, DoPont product; Mobile phase A liquid: the aqueous solution of 10mM ammonium phosphate (pH value 7.0); Mobile phase B liquid: 10mM phosphoric acid is by the aqueous solution of+60% acetonitrile, and moving phase is used preceding ultrasonic degas with 0.45 μ m membrane filtration.After the Y4 component vacuum lyophilization, dilute in right amount with mobile phase A liquid.
Method: chromatographic condition is: detect wavelength, column temperature and sampling volume with embodiment 6, and gradient elution, by the time Fractional Collections, order is compiled and is Z1, Z2, Z3, Z4, each component of ZS, Z6, and aftertreatment and determination of activity are with embodiment 6.Gradient:
Time (min) flow velocity (ml/min) B%
0.00 0.8 0
10.00 0.8 100
15.00 0.8 100
20.00 0.8 0
The results are shown in Figure 14, Figure 15, Figure 16.The relative reactivity that shows Z1, Z2, Z3, Z4, Z5, Z6 component is respectively 6.7%, 6.4%, 85.4%, 1.5% ,-6.8% ,-20.6%.Z3 component relative reactivity is higher than other component, illustrates that the Z3 component is the component that activeconstituents mainly exists.Further separate purification with the Z3 component.
The efficient positive liquid chromatography (HPNPC) of the Z3 active ingredient that embodiment 7 obtains is separated purification:
Material and instrument: high performance liquid chromatograph is the same, Zorbax RX-SIL post: 46mm * 15cm, DoPont product; Mobile phase A liquid: the aqueous solution of 0.065%TFA+10% acetonitrile (pH value 2.9), Mobile phase B liquid: the aqueous solution of 0.065%TFA+90% acetonitrile, moving phase is used preceding ultrasonic degas with 0.45 μ m membrane filtration.After the Z3 component vacuum lyophilization, with an amount of liquid dilution of mobile phase A.
Method: chromatographic condition is: detect wavelength and column temperature with embodiment 7, sampling volume: 20 μ l, gradient elution are by the time Fractional Collections, and order is compiled and is W1, W2, each component of W3, W4, and aftertreatment and active testing be with embodiment 6,7, gradient elution:
Time (min) flow velocity (ml/min) B%
0.00 0.8 0
5.00 0.8 0
10.00 0.8 10
20.00 0.8 35
30.00 0.8 100
35.00 0.8 100
45.00 0.8 0
The result sees Figure 17, Figure 18, Figure 19 respectively.The relative reactivity that shows W1, W2, W3, W4 component is respectively 6.7%, 6.4%, 50.7% ,-6.8%.W3 component relative reactivity is higher than other component, illustrates that the W3 component is the component that activeconstituents mainly exists.Select for use the W3 component further to separate purification.
Efficient positive liquid chromatography (HPNPC) separation and purification of the W3 active ingredient that embodiment 8 obtains:
Material and instrument: high performance liquid chromatograph is the same, Zorbax RX-SIL post: 4.6mm * 15cm, DoPont product; Moving phase: normal hexane/methyl alcohol=95/5,0.45 μ m membrane filtration, use preceding ultrasonic degas; After the W3 component vacuum lyophilization, with an amount of liquid dilution of moving phase.
Method: chromatographic condition is collected preceding 25% part of main peak with embodiment 8,50% part in the middle of the main peak, and 25% part and other part behind the main peak, compiling respectively is P1, P2, the p3 component, aftertreatment and survey are lived the same.
The purifying color atlas is seen Figure 20.The determination of activity result shows that the relative reactivity of P1 component is 20.9%, and the relative reactivity of P2 component is 49.6%, and the relative reactivity of P3 component is 14.7%, and the P2 component is that activeconstituents mainly exists part.
From from the raw material calf blood protein-removed extraction, each goes on foot isolating separate colors spectrogram, and promptly Fig. 5,8,11,14,17,20 carries out along with isolating as can be seen, and the purity of the component that obtains improves gradually; Fig. 5 shows a plurality of eclipsed peak, and the peak after separating in the color atlas draws back gradually, sees Fig. 8,11, obtain several comparatively clearly peaks, the quantity at peak reduces gradually, separates to final step, in the spectrogram 20 that obtains, be a main peak substantially, only contain small amount of impurities; Owing to obtain the activity of sample simultaneously with the separation and purification of Wa Shi respirometer manometry tracking and measuring, can think to have obtained a kind of bioactive peptide in the calf blood protein-removed extraction.
The blood activeconstituents, the evaluation of P2 component promptly:
1. efficient capillary zone electrophoresis (HPCZE) is identified purity:
The electrode buffer preparation of HPCZE method and pH value 2.5 is the same, and other prepares the NaH of pH value 8.0
2PO
4-Na
2HPO
4Electrode buffer: get 0.2mol/L Na
2HPO
459.2ml, 0.02mol/LNaH
2PO
43.3ml mixing is transferred pH value to 8.0, adds water to 250ml then, with 0.45 μ m membrane filtration.The dilution of P2 component is 150 μ g/ml, and sample size is 6 seconds, electrophoretic voltage 20KV.Experimental result is seen Figure 21, Figure 22.The result shows that the purity of sample reaches more than 95%.
2. high performance liquid phase gel chromatography (HPGC) determining molecular weight
The HPGC experimental technique is the same, the results are shown in Figure 23, and recording P2 component molecular weight is that 803 roads pause.
3. the column front derivation method is analyzed P2 component amino acid composition
Material and instrument: high performance liquid chromatograph is the same, Phenomonnex ODS (3) chromatography column: 4.6mm * 25cm, 17 kinds of standard amino acid solution; Concentration is about 100 μ g/ml P2 components.
Reagent: 0.1mol/L thiocarbanil (PITC) solution: get PITC12uL, add acetonitrile 988 μ l, mixing; 1mol/L triethylamine solution: get triethylamine 139 μ l, add acetonitrile 861 μ l, mixing; Mobile phase A: take by weighing the 30.3511g sodium acetate, add water 3400mL dissolving, acetate transfers PH to 6.5 to add water to 3700mL then, adds acetonitrile 300mL, adds acetone 10 μ l, uses the 0.45um membrane filtration, Mobile phase B: acetonitrile/water=4/1, use preceding ultrasonic degas.
Method: get 80 μ l P2 components and put into the sample tubule, the sample tubule is put into reaction flask, in reaction flask, add the 6NHCL solution 200 μ l that contain 1% phenol after the vacuum-drying, with air displacement is nitrogen, reaction flask is put into constent temperature heater, 150 ℃ were heated 1 hour, get 20 μ l standard amino acid solution and put into the sample tubule, reference liquid tubule and sample tubule add the solution of nitrile 10 μ l, the triethylamine 10u μ l of 100mmol/L thiocarbanil respectively, placed 1 o'clock, add 40 μ l normal hexanes respectively, mixing.Get subnatant with 0.45 μ m membrane filtration, get 1 μ l sample introduction.
Chromatographic condition is: detect wavelength 254nm, and flow velocity: 1ml/min, column temperature: 40 ℃, sampling volume: 1 μ l.
Gradient:
Time (min) flow velocity (ml/min) B%
0 1.00 0
2 1.00 5
3 1.00 7
5 1.00 12
11 1.00 15
16 1.00 30
16.01 1.00 100
20 1.00 100
20.01 1.00 0
26 1.00 0
The amino acid standard diagram is seen Figure 24, and sample hydrolysis amino acid chromatic graph spectrum is omited, and the experimental result sample is a small molecular weight titanium, and has determined its amino acid composition.
4. the ultra-violet absorption spectrum of sample
In ultrapure water, waviness is about 0.1mg/ml with the P2 components dissolved, makes blank with ultrapure water, measures the absorption spectrum of 190～300nm, and spectrogram is seen Figure 25, and as seen from Figure 25,190-220nm has intensive to absorb in the ultra-violet absorption spectrum.
5. the mass spectroscopy of sample:
The P2 component is measured through the liquid phase second order ms, has determined molecular weight and other structural information.Mass spectrum is seen Figure 26, and fragment is 589.2,545.2,403.1,346.1,244.2 and 187.2.
6. the Edman edman degradation Edman of sample order-checking:
The P2 component adopts the Edman edman degradation Edman to measure its sequence through automatic Protein Sequencer.Sequence map slightly.
In a word, the principal indication of calf blood protein-removed extraction is senile dementia and apoplexy, a large amount of basis and clinical studyes show, it is safely and effectively that deproteinized calf blood extracts these treatment of diseases, further research and discussion but the treatment mechanism of calf blood protein-removed extraction is still needed, the present invention separates the bioactive peptide and the separating and purifying method thereof of purification calf blood protein-removed extraction, is very significant for above-mentioned research and discussion.
Along with science and technology development, the discovery speed of new biological activity polypeptide increases day by day.Endocrine organ's notion has enlarged now, and the regulatory factor of a large amount of polypeptide structures is medically having potential importance.The biologically active peptides that separation and purification goes out from calf blood protein-removed extraction has the cell regulating factor possibility, is necessary to carry out further research and inquirement.
Claims (11)
1. active peptide, it is characterized in that efficient capillary zone electrophoresis evaluation purity is greater than 95%, high performance liquid phase gel chromatography molecular weight is that 803 roads pause, 190-220nm has intensive to absorb in the ultra-violet absorption spectrum, it is 589.2,545.2,403.1,346.1,244.2 and 187.2 that the liquid phase second order ms is measured fragment, Wa Shi respirometer manometry records relative reactivity 49.6%, can promote picked-up and the utilization of cavy liver cell to oxygen, be the effective constituent in the deproteinized calf blood hydrolyzed solution injection liquid.
2. the preparation method of claim 1 active peptide, it is characterized in that by active blood polypeptide through mesolow liquid chromatography, C18 and C4 reverse-phase chromatography, CN and the separation and purification of Silica normal-phase chromatography multistep, each step is measured with the relative reactivity of Wa Shi respirometer manometry tracking and measuring sample separation in the sepn process, get active maximum composition and further separate, obtain the active peptide composition of active blood.
3. preparation method according to claim 2, it is characterized in that in the sepn process of mesolow liquid chromatography, adopt the mesolow liquid chromatograph, HiLoad 16/60 Superdex post, moving phase is the aqueous solution of 10% acetonitrile+0.1%TFA, flow velocity is 1.2ml/min, and column temperature is a room temperature, and the detection wavelength is 214nm.
4. preparation method according to claim 2 is characterized in that in the efficient reversed-phase liquid chromatography sepn process, adopts high performance liquid chromatograph, Zorbax 300SB-C18 post, the aqueous solution of mobile phase A liquid: 0.1%TFA, pH value 25,0.45 μ m membrane filtration is used preceding ultrasonic degas; Mobile phase B liquid: the aqueous solution of 0.1%TFA+80% acetonitrile, detect wavelength 214nm, 37 ℃ of column temperatures, sampling volume 500 μ l, gradient elution was collected by the time,
Gradient: time (min) flow velocity (ml/min) B%
0.00 2.00 0
10.00 2.00 10
15.00 2.00 20
53.00 2.00 100
40.00 2.00 100
50.00 2.00 0
5. preparation method according to claim 2 is characterized in that adopting high performance liquid chromatograph in the efficient reversed-phase liquid chromatography sepn process, Vydac 300SB-C4 post, mobile phase A liquid: the aqueous solution of 0.1%TFA+20% acetonitrile (pH value 2.80); The acetonitrile solution of Mobile phase B liquid: 0.1%TFA detects wavelength 214nm, 37 ℃ of column temperatures, and sampling volume 50 μ 1, gradient elution is by the time Fractional Collections.
Gradient: time (min) flow velocity (ml/min) B%
0.00 1.00 0
5.00 1.00 0
15.00 1.00 55
25.00 1.00 65
30.00 1.00 65
35.00 1.00 100
40.00 1.00 100
6. preparation method according to claim 2 is characterized in that adopting high performance liquid chromatograph in efficient positive liquid chromatography (HPNPC) sepn process, Zorbax 300SB-CN post mobile phase A liquid: the aqueous solution of 10mM ammonium phosphate (pH value 7.0); Mobile phase B liquid: 10mM phosphoric acid is by the aqueous solution of+60% acetonitrile, and gradient elution is by the time Fractional Collections;
Gradient: time (min) flow velocity (ml/min) B%
0.00 0.8 0
10.00 0.8 100
15.00 0.8 100
20.00 0.8 0
7. preparation method according to claim 2, it is characterized in that adopting high performance liquid chromatograph in efficient positive liquid chromatography (HPNPC) the separation purification process, Zorbax RX-SIL post, mobile phase A liquid: the aqueous solution of 0.065%TFA+10% nitrile (pH value 2.9), Mobile phase B liquid: the aqueous solution of 0.065%TFA+90% acetonitrile, gradient elution is by the time Fractional Collections.
Gradient elution:
Time (min) flow velocity (ml/min) B%
0.00 0.8 0
5.00 0.8 0
10.00 0.8 10
20.00 0.8 35
30.00 0.8 100
35.00 0.8 100
45.00 0.8 0
8. preparation method according to claim 2 is characterized in that adopting high performance liquid chromatograph in efficient positive liquid chromatography (HPNPC) the separation and purification process, Zorbax RX-SIL post, moving phase: normal hexane/methyl alcohol=95/5.
9. active peptide according to claim 1, it is characterized in that by calf blood protein-removed extraction through mesolow liquid chromatography, C18 and C4 reverse-phase chromatography, CN and the separation and purification of Silica normal-phase chromatography multistep, each step is measured with the relative reactivity of Wa Shi respirometer manometry tracking and measuring sample separation in the sepn process, get active maximum composition and further separate, obtain the active polypeptide composition.
10. preparation method according to claim 2 is characterized in that described active blood polypeptide is a calf blood protein-removed extraction.
11. the described active peptide of claim 1 is characterized in that being used for the treatment of the cerebrovascular and wound.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100352450C (en) * | 2004-01-12 | 2007-12-05 | 江勇 | Albumen removed extraction of calf blood for injection and its preparing process |
| CN101230090B (en) * | 2007-01-24 | 2011-11-02 | 中国生化制药工业协会 | Blood peptide X and derivative thereof as well as preparation method and use thereof |
| CN111087442A (en) * | 2019-12-12 | 2020-05-01 | 石家庄高新区博科医学检验实验室有限公司 | Polypeptide extraction method |
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2000
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100352450C (en) * | 2004-01-12 | 2007-12-05 | 江勇 | Albumen removed extraction of calf blood for injection and its preparing process |
| CN101230090B (en) * | 2007-01-24 | 2011-11-02 | 中国生化制药工业协会 | Blood peptide X and derivative thereof as well as preparation method and use thereof |
| CN111087442A (en) * | 2019-12-12 | 2020-05-01 | 石家庄高新区博科医学检验实验室有限公司 | Polypeptide extraction method |
| CN111087442B (en) * | 2019-12-12 | 2024-03-12 | 石家庄博科医学检验实验室有限公司 | Polypeptide extraction method |
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