CN102399280A - Sea anemone cardiotonic peptide and extraction method and application thereof - Google Patents
Sea anemone cardiotonic peptide and extraction method and application thereof Download PDFInfo
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
An actinia cardiotonic peptide having the following amino acid sequence: GGVPC LCDSD GPSVR GNTLS GIIWL RGCPS GWHNC KAHGP TIGWC CKQ are provided. The invention also discloses an extraction method and application of the sea anemone cardiotonic peptide. Compared with the prior art, the invention has the advantages that: compared with the prior art, the invention has the advantages that: experiments on the activity mechanism of the actinocongestin AX-1 prove that the actinocongestin AX-1 has the functions of inhibiting inactivation and enhancing the sodium ion current peak value of rat myocardial cells, and is a candidate drug for treating cardiovascular diseases such as heart failure, arrhythmia and the like.
Description
Technical field
The present invention relates to a peptide species, relate in particular to a kind of cardiac stimulant peptide, the invention still further relates to the process for extracting and the application of this cardiac stimulant peptide from congestin.
Background technology
Sea anemone has another name called extra large chrysanthemum, belongs to Coelenterata (Coelenterata), Actinozoa (Anthozoa), Zoantharia (Hexacorallia), and that has reported at present surpasses 1000 kinds, and wherein China accounts for 10%.Sea anemone mainly through thread cell secretion venom predaceous fish, shellfish, copepods, Crustacean and worm, is rich in each peptide species composition in its venom, be the important content of present biotoxin research, also is one of main source of marine drug exploitation.Congestin comprises neurotoxin and cytotoxin, from about 40 kinds of sea anemones, has been separated at present to surpass 300 kinds of toxin polypeptide molecules, mainly comprises the sodium-ion channel toxin; The potassium-channel toxin; Acid-sensitive sense proton channel toxin, sea anemone synocytotoxin and enzyme inhibitors etc., wherein sea anemone sodium-ion channel toxin is that the molecule amount is the peptide molecule of 3-5kDa; Usually contain 3-4 to disulfide linkage; The sea anemone sodium-ion channel toxin of having identified at present surpasses 50 kinds, and is because sea anemone sodium-ion channel toxin specificity is strong, active special; Being research object the most interesting in the congestin, also is the main tool reagent and the potential medicine of research sodium-ion channel structure and function and sodium-ion channel relative disease.
Valtage-gated sodium-ion channel has physiological function widely, particularly in excitable cell, has mediated the granting of action potential.Being rich in the various acting peptide molecules of voltage gated ion channel that are directed against in the congestin, is the resource treasure-house of the medicine guide molecule of screening ionic channel research related tool reagent and ionic channel relative disease.The most mechanism of action of the sea anemone sodium-ion channel toxin of having identified at present is to suppress the inactivation of valtage-gated sodium-ion channel, increases sodium ion inflow, is one type of excitotoxin polypeptide.Sodium-ion channel toxin in the congestin is with its high degree of specificity and stronger activity, help to find and identify novel voltage gate sodium-ion channel albumen, analyze the proteic structure of sodium-ion channel and function, research sodium-ion channel tissue specificity, treat sodium-ion channel relative disease etc.
The congestin of at present studying at most in the world is Anthopleurin-A (Ap-A); Ap-A can be single-mindedly and irritated tissue (like neural, cardiac muscle, Skelettmuskel) cytolemma on voltage rely on the Na ionic channel and combine, its combining site is Na ionic channel 3 sites. toxin is with after the Na ionic channel combines, the opening that can prolong the Na ionic channel; Increase stream in the Na ion; The interior Na ion degree of cell is increased, and the further open and interior calcium release of stimulation through the calcium channel of Na/Ca exchanging mechanism and secondary, cause intracellular Ca ionic concn to increase; Thereby strengthen the contraction of cardiac muscle. when strengthening myocardial contraction, to heart rate, the not influence of blood pressure of laboratory animal.In addition, Ap-A also has antiarrhythmic effect, can prolong myocardial cell's Action Potential Duration, and is similar with the mechanism of action of 3 types of anti-arrhythmics, thereby Ap-A has become the polypeptide toxin that is hopeful to be developed to the cardiac stimulant peptide most.
Summary of the invention
Technical problem to be solved by this invention is to the above-mentioned state of the art a kind of new sea anemone cardiac stimulant peptide to be provided.Among the present invention with this sea anemone cardiac stimulant peptide called after AX-1.
Another technical problem to be solved by this invention provides a kind of process for extracting of sea anemone cardiac stimulant peptide.
Another technical problem to be solved by this invention provides a kind of application of sea anemone cardiac stimulant peptide.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of sea anemone cardiac stimulant peptide is characterized in that having following aminoacid sequence:
1 6 11 16 21 26 31 36 41 46
GGVPC?LCDSD?GPSVR?GNTLS?GIIWL?RGCPS?GWHNC?KAHGP?TIGWC?CKQ。
Further, comprise 6 halfcystines in this aminoacid sequence, form three pairs of disulfide linkage, the disulfide linkage mode of connection is respectively I-V, II-IV and III-VI; The molecular weight of said cardiac stimulant peptide is 5018.2Da.
Further, this cardiac stimulant peptide space structure comprises three sections antiparallel beta-pleated sheets, is respectively the peptide section that 21-24 number and 46-47 amino-acid residue constitute 3-5 number; By two big rings in three sections antiparallel beta-pleated sheet link molecule, be respectively 6-20 number and 25-45 amino-acid residue place peptide section.
A kind of process for extracting of sea anemone cardiac stimulant peptide is characterized in that comprising the steps:
1. adopt the acetone fractional precipitation method that the sea anemone crude venom is carried out preliminary extracting;
2. with preliminary extracting sample with deionized water dissolving, after the filtration, last appearance RPLC separates, and at first, adopts half preparation type C8 reversed-phase column sample to separate, and collects main peak, lyophilize;
3. utilize analysis mode C18 reversed-phase column to carry out fine separation, obtain required cardiac stimulant peptide.
Described sea anemone crude venom adopts the multigelation method from sea anemone, to extract.
Step is 1. following: acetone carries out precooling in advance under-20 ℃ of conditions, in the sea anemone crude venom, adds 20%, 50%, 80% acetone afterwards respectively and carries out fractionation precipitation, is tentatively extracted sample.
Step 2. in half preparation type C8 reversed-phase column sample to carry out sepn process following: adopt XBridgeTM C8 half preparation type reversed-phase column, elutriant is respectively water that contains 0.1%TFA and the acetonitrile that contains 0.1%TFA; Adopt linear gradient elution, acetonitrile concentration rises to 45% by 5% in 30 minutes; Flow velocity is that 2.5ml/min collects the highest main peak of ultraviolet absorption value.
Step is 3. following: adopting reverse-phase chromatographic column is 218TP54 C18, and elutriant is respectively water and the acetonitrile that contains 0.1%TFA; Adopt linear gradient elution, in the 50min, acetonitrile concentration rises to 40% from 20%, and flow velocity is 1ml/min, collects the highest main peak of ultraviolet absorption value, through freeze-dried back.
The application of a kind of sea anemone cardiac stimulant peptide in the preparation cardiovascular disease medicine.The application of a kind of sea anemone cardiac stimulant peptide in decay of preparation mental and physical efforts or cardiac arrhythmia medicine.
The evaluation of sea anemone cardiac stimulant peptide AX-1
Utilize substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF) mass spectrum (Voyager-DE
TMSTR Biospectromitry
TMWorkstation, U.S. Applied Biosystems company) measures the accurate molecular weight of toxin polypeptide.
The determined amino acid sequence of AX-1 adopts N-end Edman edman degradation Edman on the Procise 491-A of American AB I company type protein sequencer, to carry out.
The space structure of AX-1 utilizes structural simulation software ESyPred3D, since space structure (the PDB numbering: 1AHL) be template, it is carried out the space structure simulation, the main chain space structure of acquisition AX-1 of congestin Ap-A.
The active mechanism of the AX-1 of sea anemone cardiac stimulant peptide
Sodium current and calcium current record utilize magnifying glass EPC9 through full cell patch tongs technology, and (Electronid of HEKA company, Lambrecht German) carries out on computers.Computer recording and analytical system adopt Pulse+Pulsefit8.0 software.Adopt full cell patch tongs technology to detect AX-1 to the proteic inactivation of rat myocardial cell sodium-ion channel and to the influence of myocardial cell's sodium ion electric current.Series resistance is controlled at about 5M Ω, and linear capacitance electric current and leakage current subtract with P/4 program difference.All adopt in the experimentation microsyringe (IM-5B, Narishige) toxin with proper concn splashes into around experimental cell 80～100 μ m, dosing finishes, and immediately experimental cell is carried out electricity irritation and induces, and observes the influence of toxin to electric current.Adopt SigmaPlot 8.0 software analysis test-results.
Compared with prior art; The invention has the advantages that: through the experiment of sea anemone cardiac stimulant peptide AX-1 active mechanism; Proof sea anemone cardiac stimulant peptide AX-1 has the inhibition inactivation to the rat myocardial cell sodium-ion channel; Strengthening the function of sodium ion current peak, is a kind of can be used for treating heart rate depletion, drug candidate of cardiovascular disordeies such as irregular pulse.
Description of drawings
Fig. 1 is the elution curve after yellow sea anemone thick poisons half preparation type C8 reversed-phase column in Zhoushan separates behind 80% acetone precipitation.
Fig. 2 is that yellow sea anemone thick poisons C8 reversed-phase column in Zhoushan separates the elution curve of main peak after analysis mode C18 further separates of collecting the back.
Fig. 3 is that the molecular weight mass spectrum of the final sea anemone cardiac stimulant peptide AX-1 that extracts is identified figure.
Fig. 4 is the mechanism of action figure of different concns sea anemone cardiac stimulant peptide AX-1 to rat myocardial cell sodium-ion channel electric current.
Fig. 5 is not for adding AX-1 and adding behind the AX-1 rat myocardial cell sodium-ion channel electric current I-V curve comparison diagram.
Fig. 6 is not for adding AX-1 and adding behind the AX-1 rat myocardial cell sodium-ion channel electricity lead curve comparison diagram.
Embodiment
Embodiment describes in further detail the present invention below in conjunction with accompanying drawing.
The extraction of the thick poison of sea anemone:
Take out sea anemone, rinse well, insert 1000ml deionized water (containing 15ml 0.5M EDTA, 300 μ L2mg/ml Trypsin inhibitor,Trasylols) with deionized water, freezing under-80 ℃ of conditions rapidly behind the mixing; After freezing 12 hours, take out the refrigerated sea anemone, dissolving at room temperature, treat that freezing sea anemone is all melted after, the sea anemone sample is placed on once more in-80 ℃ the refrigerated tank freezing 12 hours; Take out refrigerated sea anemone sample afterwards, place under the room temperature and fully melt, above-mentioned steps repeats 2-3 time; Sea anemone sample after melting is carried out coarse filtration with the gauze parcel to it, remove impurity such as sea anemone body, get its filtrating; Filtrating is placed high speed freezing centrifuge, and under 4 ℃, 20000 * g condition, centrifugal 20min gets its supernatant; After repeating to filter twice, supernatant is subsequent use, is the thick malicious sample of sea anemone, places 4 ℃ of preservations subsequent use.
Adopt the acetone fractional precipitation method that the sea anemone crude venom is carried out preliminary extracting.Acetone carries out precooling in advance under-20 ℃ of conditions; In the sea anemone crude venom, add 20%, 50%, 80% acetone afterwards respectively and carry out fractionation precipitation; Each step gained is deposited under 4 ℃, 15000 * g condition; Centrifugal 10min, the gained deposition is collected postlyophilization, is stored in-20 ℃ of refrigerators subsequent use.
The extraction of sea anemone cardiac stimulant peptide AX-1:
Emphasis carries out further separation and purification to the thick poisons 80% acetone precipitation gained sample of sea anemone.Deposit sample is with deionized water dissolving, and after 0.45m aperture filter filtered, last appearance RPLC separated.All separating steps are all accomplished on Waters 600E type high performance liquid chromatograph, and detector is Waters 2487 type UV-detectors, and the detection wavelength is 280nm.
The first step: adopt XBridgeTM C8 half preparation type reversed-phase column (10*250mm, U.S. waters company), elutriant is respectively water (A liquid) that contains 0.1%TFA (v/v) and the acetonitrile (B liquid) that contains 0.1%TFA (v/v); Adopt linear gradient elution, B liquid concentration rises to 45% by 5% in 30 minutes; Flow velocity is that 2.5ml/min collects the highest main peak of ultraviolet absorption value, is placed on through lyophilize and preserves subsequent usely in the refrigerator, sees shown in the accompanying drawing 1, is labeled as collected main peak " * " among the figure number.
Second step: carry out further RPLC separation to collecting the highest main peak composition of ultraviolet absorption value in the above-mentioned steps; Reverse-phase chromatographic column is 218TP54 C18 (4.6 * 250mm; Vydac company), elutriant is respectively water (A liquid) and the acetonitrile (B liquid) that contains 0.1%TFA; Adopt linear gradient elution, in the 50min, B liquid rises to 40% from 20%, and flow velocity is 1ml/min, collects the highest main peak of ultraviolet absorption value, after lyophilize, identifies, sees shown in the accompanying drawing 2, is labeled as collected main peak " * " among the figure number.
The evaluation of congestin AX-1:
Utilize substance assistant laser desorpted ionized flight time mass spectrum (MALDI-TOF) mass spectrum (Voyager-DETMSTRBiospectromitry
TMWorkstation, U.S. Applied Biosystems company) measures the accurate molecular weight of toxin polypeptide.Adopt linear cation mode; N
2Light source 337nm; Ion-accelerating voltage is 20000V.Matrix is alpha-cyano-4-hydroxyl-styracin (α-cyano-4-hydroxy-cinnamic acid; CCA); Prepare sample in the following manner: get 50% acetonitrile saturated solution (containing 0.1%TFA) that 1 μ L sample liquid joins 9 μ L CCA; Get 1 μ L point sample behind the mixing, carry out mass spectroscopy after the drying at room temperature, molecular weight is proofreaied and correct with marker method.As shown in Figure 3, mass spectrum is the result show, the accurate molecular weight of the Zhoushan yellow sea anemone cardiac stimulant peptide AX-1 that is extracted is 5018.2Da, and purity is higher.
The determined amino acid sequence of AX-1 adopts N-end Edman edman degradation Edman on the Procise 491-A of American AB I company type protein sequencer, to carry out.Adopt the standard program order-checking of apparatus preparation, survey 50 circulations, online RPLC detects and combines the accurate collection of illustrative plates of amino acidity scale to judge the amino acid kind, finally accurately reads the aminoacid sequence of institute's test sample article.The sequencing results shows that AX-1 is made up of 48 amino-acid residues, and its sequence is following:
GGVPCLCDSDGPSVRGNTLSGIIWLRGCPSGWHNCKAHGP TIGWCCKQ; Comprise six halfcystines in the sequence,, judge that AX-1 forms three groups of disulfide linkage according to mass spectrum molecular weight and theoretical molecular difference.
The space structure of congestin polypeptide utilizes structural simulation software ESyPred3D; With space structure (PDB numbering: 1AHL) be template from the toxin A p-A of yellow sea anemone; It is carried out the space structure simulation, obtain the main chain space structure of Zhoushan yellow sea anemone toxin polypeptide.Structural analysis shows that AX-1 has taked the similar backbone structure with Ap-A, comprises three sections antiparallel beta-pleated sheets in its structure, is respectively the peptide section that 21-24 number and 46-47 amino-acid residue constitute 3-5 number; By two the big rings (loop) in three sections antiparallel beta-pleated sheet link molecule, be respectively 6-20 number and 25-45 amino-acid residue place peptide section.The distribution of four key amino acid residues and Ap-A are similar in its molecule, by Asp8, and Asp10, Lys36, the side chain of His38 is spatially assembled distribution, forms the avtive spot of AX-1.
The active mechanism of sea anemone cardiac stimulant peptide AX-1:
The sodium current record utilizes magnifying glass EPC9 through full cell patch tongs technology, and (Electronid of HEKA company, Lambrecht German) carries out on computers.Computer recording and analytical system adopt Pulse+Pulsefit 8.0 softwares.The glass electrode pipe is 100 μ L (VWR micropipettes, VWR Company) borosilicate glass capillary tube.Two steps of glass electrode draw and form, and (Narishige Japan) polishes the rear electrode tip diameter and is about 3 μ m, and charging electrode solution rear electrode resistance is 1-3M Ω through the polishing appearance.Patch clamp experiments will be carried out at ambient temperature, and the change of temperature is no more than 2 ℃ up and down in the whole experiment.
The extracellular fluid (mM of unit) of record rat myocardial cell sodium current: NaCl 145, and KCl 5.5, MgCl
21, CdCl
22, glucose 10, and HERES 10, with 1MNaOH and 1MHCl the pH value transferred to 7.4; Liquid (mM) in the electrode: KCl155, MgCl
21, Na
2-ATP 3, Tris phosphocreatine 5, and HERES 10, with 1M KOH and 1M HCl the pH value transferred to 7.4.
Above intracellular fluid and outer liquid are all through 0.22 μ m membrane filtration.Under the room temperature; Select suitable cell under the inverted microscope; When clamping down on after cell has formed high resistant (1-5G) in little Glass tubing sealing-in, automatically perform the C-fast capacitance compensation, clamp down in advance in-60mV; Further the suction microelectrode is clamped down on logging mode (Whole-cell patchclamp) thereby make membranolysis form full cell.Automatically perform C-slow capacitance compensation and series resistance (R series) compensation, clamp down on, form voltage clamp in the target current potential, membrane current application 1 0KHz filtering, stablize 4-6min after, apply the depolarize pulse, the TCH test channel current conditions.Patch clamp amplifier is that (List-Electronic German), uses Pulse/Pulsefit 8.0 softwares (HEKA Electronics, Germany) Collection and analysis record data to the EPC-9 type.Series resistance is controlled at about 5M Ω, and linear capacitance electric current and leakage current subtract with P/4 program difference.All adopt in the experimentation microsyringe (IM-5B, Narishige) toxin with proper concn splashes into around experimental cell 80～100 μ m, dosing finishes, and immediately experimental cell is carried out electricity irritation and induces, and observes the influence of toxin to electric current.Adopt SigmaPlot 8.0 software analysis test-results.
Congestin AX-1 is to the effect of rat drg neuron sodium channel.Cell membrane potential is clamped down at-80mV, given one TV-10mV, whenever repeat once at a distance from 5s with the time-histories of 50ms.In conjunction with shown in Figure 4,1 μ M, 10 μ MAX-1 all can increase sodium channel current, also can delay the inactivation of sodium-ion channel simultaneously.1 μ MAX-1 can increase about 149.29 ± 35.24%, the 10 μ M AX-1 of the sodium channel current of rat myocardial cell, and to increase sodium channel current about 340.73 ± 29.79%, also can obviously delay the rapid deactivation time of sodium channel simultaneously.Under the blank condition; Sodium current inactivation almost completely when depolarize 5ms, but after adding toxin, sodium current inactivation not during depolarize 5ms; This shows that congestin AX-1 can not only increase the sodium current intensity on the rat myocardial cell, can delay the inactivation of sodium channel simultaneously.
Congestin AX-1 is to the sodium channel kinetic effect.Cell command potential-80mV, the TV variation range from-80mV～+ 50mV, TV time length 50ms, transition stride+10mV.Fig. 5 makes the I-V curve of sodium channel toward the drift of hyperpolarization direction for I-V curvilinear motion after not adding toxin and adding toxin, 10 μ M toxin.Fig. 6 changes for electric lead curve after not adding toxin and adding toxin, and 10 μ M toxin make the electric lead curve of sodium channel toward the drift of hyperpolarization direction.Under full cell pattern, detect AX-1 the myocardial cell is gone up sodium-ion channel current-voltage curve influence (I-V), with the influence of judging that toxin is open to sodium-ion channel.Cell command potential-80mV, the TV variation range from-80mV～+ 50mV, TV time length 50ms, transition stride+10mV.Do not adding under the toxin collating condition, the initial activation voltage of myocardium sodium channel is-30mV, and the peak inrush current activation voltage is-10mV, reversal potential is+25mV about.After adding 10 μ M toxin; The initial activation voltage of sodium channel is-40mV; The peak inrush current activation voltage is-20mV that the toxin of 10 μ M makes sodium-ion channel I-V curve toward hyperpolarization direction drift 10mV, also increases considerably sodium channel I-V peak of curve current amplitude simultaneously.The electricity lead curve shows that equally 10 μ MAX-1 toxin can make the activation of sodium channel toward the drift of hyperpolarization direction.
Zhoushan yellow sea anemone in the present embodiment distributes extensively, and material is easy to get, and the process for extracting that utilizes present embodiment to provide can extract about 20 milligrams of AX-1 from one kilogram of sea anemone.
Claims (10)
1. sea anemone cardiac stimulant peptide is characterized in that having following aminoacid sequence:
1 6 11 16 21 26 31 36 41 46
GGVPC?LCDSD?GPSVR?GNTLS?GIIWL?RGCPS?GWHNC?KAHGP?TIGWC?CKQ。
2. sea anemone cardiac stimulant peptide according to claim 1 is characterized in that comprising in this aminoacid sequence 6 halfcystines, forms three pairs of disulfide linkage, and the disulfide linkage mode of connection is respectively I-V, II-IV and III-VI; The molecular weight of said cardiac stimulant peptide is 5018.2Da.
3. sea anemone cardiac stimulant peptide according to claim 2 is characterized in that this cardiac stimulant peptide space structure comprises three sections antiparallel beta-pleated sheets, is respectively the peptide section that 21-24 number and 46-47 amino-acid residue constitute 3-5 number; By two big rings in three sections antiparallel beta-pleated sheet link molecule, be respectively 6-20 number and 25-45 amino-acid residue place peptide section.
4. the process for extracting of any one sea anemone cardiac stimulant peptide in the claim 1～3 is characterized in that comprising the steps:
1. adopt the acetone fractional precipitation method that the sea anemone crude venom is carried out preliminary extracting;
2. with preliminary extracting sample with deionized water dissolving, after the filtration, last appearance RPLC separates, and at first, adopts half preparation type C8 reversed-phase column sample to separate, and collects main peak, lyophilize;
3. utilize analysis mode C18 reversed-phase column to carry out fine separation, obtain required cardiac stimulant peptide.
5. process for extracting according to claim 4 is characterized in that described sea anemone crude venom adopts the multigelation method from sea anemone, to extract.
6. process for extracting according to claim 4, it is characterized in that step is 1. following: acetone carries out precooling in advance under-20 ℃ of conditions, in the sea anemone crude venom, adds 20%, 50%, 80% acetone afterwards respectively and carries out fractionation precipitation, is tentatively extracted sample.
7. process for extracting according to claim 4; It is following to it is characterized in that during step 2. that half preparation type C8 reversed-phase column sample carries out sepn process: adopt XBridgeTM C8 half preparation type reversed-phase column, elutriant is respectively water that contains 0.1%TFA and the acetonitrile that contains 0.1%TFA; Adopt linear gradient elution, acetonitrile concentration rises to 45% by 5% in 30 minutes; Flow velocity is that 2.5ml/min collects the highest main peak of ultraviolet absorption value.
8. process for extracting according to claim 4, it is characterized in that step is 3. following: the employing reverse-phase chromatographic column is 218TP54C18, elutriant is respectively water and the acetonitrile that contains 0.1%TFA; Adopt linear gradient elution, in the 50min, acetonitrile concentration rises to 40% from 20%, and flow velocity is 1ml/min, collects the highest main peak of ultraviolet absorption value, through freeze-dried back.
9. the application of any one sea anemone cardiac stimulant peptide in the preparation cardiovascular disease medicine in the claim 1～3.
10. the application of any one sea anemone cardiac stimulant peptide in decay of preparation mental and physical efforts or cardiac arrhythmia medicine in the claim 1～3.
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| CN108478781A (en) * | 2018-05-08 | 2018-09-04 | 大连理工大学 | The lyophilized technique of injection cardiac muscle peptide |
| CN108794578A (en) * | 2018-07-03 | 2018-11-13 | 宋雪萍 | A kind of preparation method and its pharmaceutical composition of octreotide acetate |
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| CN103205350A (en) * | 2012-12-21 | 2013-07-17 | 江苏省海洋水产研究所 | Preparation method of sea anemone wine |
| CN104231105A (en) * | 2014-09-28 | 2014-12-24 | 浙江工业大学 | Preparation method for sea anemone polysaccharide and application thereof to tumor resistance |
| CN104231105B (en) * | 2014-09-28 | 2016-06-22 | 浙江工业大学 | The preparation method of a kind of sea anemone polysaccharide and antitumor application thereof |
| CN105030838A (en) * | 2015-05-31 | 2015-11-11 | 浙江海洋学院 | Preparing method of sea anemone crude extract and anti-tumor application of sea anemone crude extract |
| CN108478781A (en) * | 2018-05-08 | 2018-09-04 | 大连理工大学 | The lyophilized technique of injection cardiac muscle peptide |
| CN108794578A (en) * | 2018-07-03 | 2018-11-13 | 宋雪萍 | A kind of preparation method and its pharmaceutical composition of octreotide acetate |
| CN108794578B (en) * | 2018-07-03 | 2020-06-02 | 北京市新里程医药科技有限公司 | Preparation method of octreotide acetate and pharmaceutical composition thereof |
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