CN108148111A - The extracting method of polypeptide in a kind of spleen aminopeptide - Google Patents
The extracting method of polypeptide in a kind of spleen aminopeptide Download PDFInfo
- Publication number
- CN108148111A CN108148111A CN201711375642.XA CN201711375642A CN108148111A CN 108148111 A CN108148111 A CN 108148111A CN 201711375642 A CN201711375642 A CN 201711375642A CN 108148111 A CN108148111 A CN 108148111A
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- Prior art keywords
- mobile phase
- polypeptide
- spleen aminopeptide
- spleen
- aminopeptide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
Abstract
The invention discloses a kind of extracting methods of polypeptide in spleen aminopeptide, include the following steps, step 1, preparation of reagents, prepare mobile phase A, Mobile phase B；Spleen aminopeptide is added in starting mobile phase, obtains sample solution by step 2, step 2, sample treatment；Step 3: gradient elution.The present invention has the following advantages and effects：Using the polypeptide in chromatographic isolation extraction spleen aminopeptide, during sample treatment, with 0.22 μm of organic film filtered sample, the macromolecular substances in spleen aminopeptide sample are removed, interference when reducing to extraction polypeptide；Secondly, gradient elution is carried out after sample introduction, by adjusting the ratio between mobile phase A and Mobile phase B so that polypeptide is concentrated at main chromatographic peak and eluted, and has been reached the polypeptide in extraction spleen aminopeptide, has been isolated and purified the good effect of effect.
Description
Technical field
The present invention relates to field of biological product, the extracting method of polypeptide in more particularly to a kind of spleen aminopeptide.
Background technology
The oral freeze-dried powder of spleen aminopeptide belongs to the Biochemical Drugs extracted from internal organs, and main component includes amino acid, polypeptide, nucleotide
Substances are waited, as immune body conditioning agent, for the auxiliary of the illnesss such as repeated respiratory infections, bronchitis, asthma, pneumonia, cancer
Help treatment.Clinical observation is shown, can effectively improve the resistance of patient, reduces the incidence of respiratory tract infection, and with tune
The function that ganglion cell is immunized, main active material is polypeptide in spleen aminopeptide, national drug standards WS1- (X-002) -2002Z
In, the content for measuring polypeptide is to control the sole indicator of spleen aminopeptide product content, by the peptide separation in spleen aminopeptide after purification, right
In research spleen aminopeptide as the mechanism of action of immunomodulator and using the polypeptide of separation as freeze-dried powder preparation to significantly improve
The vigor of drug, all with obvious action and meaning, therefore, it is necessary to propose a kind of extracting method of polypeptide in spleen aminopeptide.
Invention content
The object of the present invention is to provide a kind of extracting methods of polypeptide in spleen aminopeptide, have the effect of polypeptide in separation spleen aminopeptide
Fruit.
The present invention above-mentioned technical purpose technical scheme is that：Polypeptide carries in a kind of spleen aminopeptide
Method is taken, is included the following steps, step 1, preparation of reagents prepares mobile phase A, Mobile phase B, and mobile phase A internal solvent is water, molten
Matter is the formic acid that mass percent concentration is 0.1, and Mobile phase B internal solvent is acetonitrile, solute is including mass percent concentration
0.1% formic acid and 0.05% adipic acid；Spleen aminopeptide is added in starting mobile phase, obtains sample by step 2, sample treatment
Product solution；Step 3 isolates and purifies, chromatographic condition, and chromatographic column filler is Bio-sil, a diameter of 5 μm of filler particles, chromatographic column
Internal diameter 2.1mm, column length 75mm, 40 DEG C, gradient elution 0min-5min-14min-18min-24.5min-30min of column temperature, flowing
Phase B 5%-12%-17%-17.5%-30%-50%；Flow velocity is 0.5mL/min, and sampling volume is 20 μ L, Detection wavelength
214nm collects main chromatographic peak.
By using above-mentioned technical proposal, mainly include the components such as polypeptide, nucleotide, free amino acid, group in spleen aminopeptide
Divide complexity, each component molecular size range differs.The application uses chromatographic separation technology, and chromatographic isolation is a kind of separation COMPLEX MIXED
The effective ways of various components in object have different distribution using different material in the system of stationary phase and mobile phase composition
Coefficient, when mobile phase and stationary phase relative motion, these substances are moved with mobile phase, and in two alternate progress repeatedly
Distribution, so as to which each component be made to reach separation.
In chromatographic separation condition, can the selection of mobile phase divide out of spleen aminopeptide obtain suitable peak type, success
Separate out that polypeptide is most important, select the mode of gradient elution, the aqueous solution of mobile phase A formic acid, Mobile phase B include acetonitrile,
Formic acid and adipic acid, acetonitrile, formic acid are common mobile phase composition in chromatographic isolation, and the application adds in oneself two in Mobile phase B
Acid, after being eluted by above-mentioned condition of gradient elution, when elution, can will elute in the polypeptide set in spleen aminopeptide, concentrate on main
At chromatographic peak, the peptide separation in spleen aminopeptide is come out, good separating effect.
The further setting of the present invention is：Step 2, sample treatment take spleen aminopeptide stoste to centrifuge, and centrifugal condition is：
12000rpm, 10min, 4 DEG C, supernatant is taken after centrifugation, is added in starting mobile phase, is filtered, obtained with 0.22 μm of organic film
Sample solution.
By using above-mentioned technical proposal, during sample treatment, spleen aminopeptide stoste is taken to centrifuge, is centrifuged 10 minutes at 4 DEG C, then
It is filtered by the organic film that aperture is 0.22 μm, removes the big molecular impurity in spleen aminopeptide stoste, divide greatly when reducing chromatographic isolation
The interference of sub- substance improves the accuracy of chromatographic isolation extraction polypeptide.
The further setting of the present invention is：In step 2, every 600 μ L supernatants add in 100 μ L starting mobile phases.
By using above-mentioned technical proposal, every 600 μ L supernatants add in 100 μ L starting mobile phases, and starting mobile phase refers to
During chromatographic isolation gradient elution, the mobile phase of starting, the chromatographic condition in step 3 is it is found that starting mobile phase includes 95%
Mobile phase A and 5% Mobile phase B.
The further setting of the present invention is：Step 2, sample treatment take spleen aminopeptide freeze-dried powder to be dissolved in deionized water, 4 DEG C
It is lower to be dialysed with bag filter, starting mobile phase is added in the spleen aminopeptide stoste after dialysis, is filtered, obtained with 0.22 μm of organic film
Sample solution.
By using above-mentioned technical proposal, when spleen aminopeptide is freeze-dried powder, spleen aminopeptide freeze-dried powder is taken to be dissolved in deionized water,
It is dialysed with bag filter, the molecular cut off of bag filter can be selected according to the size of target polypeptides, and backward spleen aminopeptide of dialysing is former
Starting mobile phase is added in liquid, after being filtered by organic film, obtains sample solution.
The further setting of the present invention is：In step 2, every 500 μ L spleen aminopeptides stoste adds in 200 μ L starting mobile phases.
By using above-mentioned technical proposal, every 500 μ L spleen aminopeptides stoste adds in 200 μ L starting mobile phases.
The further setting of the present invention is：Step 3 in isolating and purifying, collects 11.222-11.711min, 25.719-
25.919min、25.935-26.285min、26.302-26.652min、30.016-30.240min、30,999-31.049min
Chromatographic peak.
By using above-mentioned technical proposal, under the chromatographic condition of step 3, main chromatographic peak includes above-mentioned 6 time
Section, the polypeptide in spleen aminopeptide is concentrated in above-mentioned 6 chromatographic peaks, collects the polypeptide that can be detached.
In conclusion the invention has the advantages that：Using the polypeptide in chromatographic isolation extraction spleen aminopeptide, at sample
During reason, with 0.22 μm of organic film filtered sample, the macromolecular substances in spleen aminopeptide sample are removed, when reducing to extraction polypeptide
Interference；Secondly, gradient elution is carried out after sample introduction, by adjusting the ratio between mobile phase A and Mobile phase B so that in polypeptide set
It is eluted at main chromatographic peak, has reached the polypeptide in extraction spleen aminopeptide, isolated and purified the good effect of effect.
Specific embodiment
Embodiment 1：The extracting method of polypeptide, includes the following steps in a kind of spleen aminopeptide, step 1, preparation of reagents, prepares
Mobile phase A, Mobile phase B, mobile phase A internal solvent is water, solute is formic acid that mass percent concentration is 0.1, molten in Mobile phase B
The formic acid and 0.05% adipic acid that agent is acetonitrile, solute is 0.1% including mass percent concentration.
Step 2, sample treatment take spleen aminopeptide stoste to centrifuge, and centrifugal condition is：12000rpm, 10min, 4 DEG C, after centrifugation
Supernatant is taken, is added in starting mobile phase, every 600 μ L supernatants add in 100 μ L starting mobile phases, with 0.22 μm of organic membrane filtration
It crosses, obtains sample solution.Wherein, by volume, starting mobile phase includes 95% mobile phase A and 5% Mobile phase B.
Step 3 isolates and purifies, chromatographic condition, and chromatographic column filler is Bio-sil, a diameter of 5 μm of filler particles, chromatography
Column internal diameter 2.1mm, column length 75mm, chromatographic column select Agilent Poroshell 300SB-C18.40 DEG C of column temperature, gradient elution
0min-5min-14min-18min-24.5min-30min, Mobile phase B 5%-12%-17%-17.5%-30%-50%；Stream
Speed is 0.5mL/min, and sampling volume is 20 μ L, Detection wavelength 214nm, collects 11.222-11.711min, 25.719-
25.919min、25.935-26.285min、26.302-26.652min、30.016-30.240min、30,999-31.049min
Chromatographic peak.
The sample of each period is collected, nucleotide content, sugared content, content of peptides in each sample is detected, is arranged in table 2
Go out testing result.
Embodiment 2：The extracting method of polypeptide in a kind of spleen aminopeptide, the difference lies in, step 2, samples with embodiment 1
Product processing, takes spleen aminopeptide freeze-dried powder to be dissolved in deionized water, is dialysed at 4 DEG C with bag filter, in the spleen aminopeptide stoste after dialysis
Starting mobile phase is added in, every 500 μ L spleen aminopeptides stoste adds in 200 μ L starting mobile phases, filtered, obtained with 0.22 μm of organic film
Sample solution.
Table 1
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 5 | 88 | 12 |
| 14 | 83 | 17 |
| 18 | 82.5 | 17.5 |
| 24.5 | 70 | 30 |
| 30 | 50 | 50 |
Table 2
| Sample name | Period/min | Nucleotide (μ g/ml) | Sugared (μ g/ml) | Polypeptide (μ g/ml) |
| Fraction1 | 0-11.222 | ＞ 15 | 2.5-5 | 0.025-0.05 |
| Fraction2 | 11.222-11.711 | 8-12 | 2.5-5 | 0.4-0.5 |
| Fraction3 | 11.711-25.719 | 8-12 | 2.5-5 | 0.1-0.2 |
| Fraction4 | 25.719-25.919 | Nothing | 1.2-2 | 0.4-0.5 |
| Fraction5 | 25.935-26.285 | Nothing | 1.2-2 | 0.4-0.5 |
| Fraction6 | 26.302-26.652 | Nothing | 1.2-2 | 0.4-0.5 |
| Fraction7 | 26.652-30.016 | Nothing | 1.2-2 | 0.1-0.2 |
| Fraction8 | 30.016-30.240 | Nothing | 1.2-2 | 0.4-0.5 |
| Fraction9 | 30.999-31.049 | Nothing | 1.2-2 | 0.4-0.5 |
Comparative example：It is in place of the extracting method of polypeptide in a kind of spleen aminopeptide, with the difference of embodiment 1, it is molten in Mobile phase B
The formic acid that agent is acetonitrile, solute is 0.1% including mass percent concentration.
The sample of each period is collected, nucleotide content, sugared content, content of peptides in each sample is detected, is arranged in table 3
Go out testing result.
Table 3
| Sample name | Period/min | Nucleotide (μ g/ml) | Sugared (μ g/ml) | Polypeptide (μ g/ml) |
| Fraction1 | 0-11.222 | ＞ 15 | 2.5-5 | 0.025-0.05 |
| Fraction2 | 11.222-11.711 | 8-12 | 2.5-5 | 0.2-0.3 |
| Fraction3 | 11.711-25.719 | 4-5 | 2.5-5 | 0.2-0.3 |
| Fraction4 | 25.719-25.919 | 4-5 | 2.5-5 | 0.2-0.3 |
| Fraction5 | 25.935-26.285 | 4-5 | 1-2 | 0.2-0.3 |
| Fraction6 | 26.302-26.652 | 4-5 | 1-2 | 0.2-0.3 |
| Fraction7 | 26.652-30.016 | 4-5 | Nothing | 0.2-0.3 |
| Fraction8 | 30.016-30.240 | 4-5 | Nothing | 0.2-0.3 |
| Fraction9 | 30.999-31.049 | 4-5 | Nothing | 0.05-0.1 |
Specific embodiment is only explanation of the invention, is not limitation of the present invention, those skilled in the art
The modification of no creative contribution can be made to the present embodiment as needed after this specification is read, but as long as in this hair
It is all protected in bright right by Patent Law.
Claims (6)
1. a kind of extracting method of polypeptide in spleen aminopeptide, it is characterised in that：Include the following steps
Step 1, preparation of reagents prepare mobile phase A, Mobile phase B, and mobile phase A internal solvent is water, solute is that mass percent is dense
Spend the formic acid for 0.1, the formic acid and 0.05% that Mobile phase B internal solvent is acetonitrile, solute is 0.1% including mass percent concentration
Adipic acid；
Spleen aminopeptide is added in starting mobile phase, obtains sample solution by step 2, sample treatment；
Step 3 isolates and purifies, chromatographic condition, and chromatographic column filler is Bio-sil, a diameter of 5 μm of filler particles, in chromatographic column
Diameter 2.1mm, column length 75mm, 40 DEG C, gradient elution 0min-5min-14min-18min-24.5min-30min of column temperature, Mobile phase B
5%-12%-17%-17.5%-30%-50%；Flow velocity is 0.5mL/min, and sampling volume is 20 μ L, Detection wavelength 214nm,
Collect main chromatographic peak.
2. the extracting method of polypeptide in a kind of spleen aminopeptide according to claim 1, it is characterised in that：Step 2, at sample
Reason, takes spleen aminopeptide stoste to centrifuge, and centrifugal condition is：12000rpm, 10min, 4 DEG C, supernatant is taken after centrifugation, be added to starting stream
In dynamic phase, filtered with 0.22 μm of organic film, obtain sample solution.
3. the extracting method of polypeptide in a kind of spleen aminopeptide according to claim 2, it is characterised in that：In step 2, every 600
μ L supernatants add in 100 μ L starting mobile phases.
4. the extracting method of polypeptide in a kind of spleen aminopeptide according to claim 1, it is characterised in that：Step 2, at sample
Reason, takes spleen aminopeptide freeze-dried powder to be dissolved in deionized water, is dialysed at 4 DEG C with bag filter, added in the spleen aminopeptide stoste after dialysis
Mobile phase is originated, is filtered with 0.22 μm of organic film, obtains sample solution.
5. the extracting method of polypeptide in a kind of spleen aminopeptide according to claim 4, it is characterised in that：In step 2, every 500
μ L spleen aminopeptides stoste adds in 200 μ L starting mobile phases.
6. the extracting method of polypeptide in a kind of spleen aminopeptide according to claim 1, it is characterised in that：Step 3, separation are pure
In change, collect 11.222-11.711min, 25.719-25.919min, 25.935-26.285min, 26.302-26.652min,
30.016-30.240min, the chromatographic peak of 30,999-31.049min.
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| CN201711375642.XA CN108148111B (en) | 2017-12-19 | 2017-12-19 | Method for extracting polypeptide in spleen aminopeptide |
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| CN201711375642.XA CN108148111B (en) | 2017-12-19 | 2017-12-19 | Method for extracting polypeptide in spleen aminopeptide |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113797756A (en) * | 2021-09-08 | 2021-12-17 | 北京腾跃高科科技有限公司 | Separation, purification and extraction equipment for spleen peptide |
| CN114940698A (en) * | 2022-07-22 | 2022-08-26 | 北京第一生物化学药业有限公司 | Method for extracting acidic polypeptide from spleen aminopeptide stock solution |
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| RU2033796C1 (en) * | 1993-07-16 | 1995-04-30 | Чернов Василий Александрович | Peptide-containing fraction from mammalian spleen showing immunostimulating activity, and a method of its preparing |
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| US20080319163A1 (en) * | 2005-04-21 | 2008-12-25 | Yuliang Wang | Method for Isolating and Purifying Immuno-Modulating Polypeptide from Cow Placenta |
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2017
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| RU2033796C1 (en) * | 1993-07-16 | 1995-04-30 | Чернов Василий Александрович | Peptide-containing fraction from mammalian spleen showing immunostimulating activity, and a method of its preparing |
| US20080319163A1 (en) * | 2005-04-21 | 2008-12-25 | Yuliang Wang | Method for Isolating and Purifying Immuno-Modulating Polypeptide from Cow Placenta |
| CN101254209A (en) * | 2008-03-26 | 2008-09-03 | 吉林敖东洮南药业股份有限公司 | Calf spleen extract injection prepared by inphase opposition column chromatography and preparation thereof |
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Non-Patent Citations (2)
| Title |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113797756A (en) * | 2021-09-08 | 2021-12-17 | 北京腾跃高科科技有限公司 | Separation, purification and extraction equipment for spleen peptide |
| CN113797756B (en) * | 2021-09-08 | 2024-02-02 | 北京腾跃高科科技有限公司 | Separation and purification extraction equipment of spleen peptide |
| CN114940698A (en) * | 2022-07-22 | 2022-08-26 | 北京第一生物化学药业有限公司 | Method for extracting acidic polypeptide from spleen aminopeptide stock solution |
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