CN108148111B - Method for extracting polypeptide in spleen aminopeptide - Google Patents
Method for extracting polypeptide in spleen aminopeptide Download PDFInfo
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- CN108148111B CN108148111B CN201711375642.XA CN201711375642A CN108148111B CN 108148111 B CN108148111 B CN 108148111B CN 201711375642 A CN201711375642 A CN 201711375642A CN 108148111 B CN108148111 B CN 108148111B
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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
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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
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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Abstract
The invention discloses a method for extracting polypeptide in spleen aminopeptide, which comprises the following steps of preparing a reagent, and preparing a mobile phase A and a mobile phase B; step two, sample treatment, namely adding spleen aminopeptide into the initial mobile phase to obtain a sample solution; step three, gradient elution. The invention has the following advantages and effects: the polypeptide in the spleen aminopeptide is separated and extracted by adopting a chromatograph, and when a sample is treated, the sample is filtered by using an organic membrane with the diameter of 0.22 mu m, so that macromolecular substances in the spleen aminopeptide sample are removed, and the interference on the extraction of the polypeptide is reduced; secondly, after sample injection, gradient elution is carried out, and the polypeptide is eluted at the main chromatographic peak in a centralized manner by adjusting the proportion between the mobile phase A and the mobile phase B, so that the effects of extracting the polypeptide in the spleen aminopeptide and having good separation and purification effects are achieved.
Description
Technical Field
The invention relates to the field of biological products, in particular to a method for extracting polypeptide in spleen aminopeptide.
Background
The spleen aminopeptide freeze-dried oral powder is a biochemical medicine extracted from internal organs, and its main component includes amino acids, polypeptide and nucleotide, etc. and can be used as immune body regulator for auxiliary treatment of diseases of repeated respiratory tract infection, bronchitis, asthma, pneumonia and cancer, etc. Clinical observation shows that the spleen aminopeptide has the functions of effectively improving the resistance of patients, reducing the incidence rate of respiratory tract infection and regulating cellular immunity, the main active substance in the spleen aminopeptide is polypeptide, the content of the polypeptide is determined to be the only index for controlling the content of the spleen aminopeptide product in the national drug standard WS1- (X-002) -2002Z, and the polypeptide in the spleen aminopeptide is separated and purified to have obvious action and significance for researching the action mechanism of the spleen aminopeptide as an immunomodulator and obviously improving the activity of the drug by taking the separated polypeptide as a freeze-dried powder preparation, so that the method for extracting the polypeptide in the spleen aminopeptide is needed to be provided.
Disclosure of Invention
The invention aims to provide an extraction method of polypeptide in spleen aminopeptide, which has the effect of separating the polypeptide in the spleen aminopeptide.
The technical purpose of the invention is realized by the following technical scheme: a method for extracting polypeptide in spleen aminopeptide comprises the following steps of preparing a reagent, preparing a mobile phase A and a mobile phase B, wherein a solvent in the mobile phase A is water, a solute is formic acid with the mass percent concentration of 0.1, a solvent in the mobile phase B is acetonitrile, and the solute comprises formic acid with the mass percent concentration of 0.1% and adipic acid with the mass percent concentration of 0.05%; step two, sample treatment, namely adding spleen aminopeptide into the initial mobile phase to obtain a sample solution; step three, separating and purifying, wherein the chromatographic conditions are that the chromatographic column filler is porous silica gel, the diameter of the filler particles is 5 microns, the inner diameter of the chromatographic column is 2.1mm, the length of the chromatographic column is 75mm, the Agilent Poroshell 300SB-C18 is selected as the chromatographic column, the temperature of the chromatographic column is 40 ℃, gradient elution is carried out for 0min-5min-14min-18min-24.5min-30min, and the flow B5% -12% -17% -17.5% -30% -50%; the flow rate is 0.5mL/min, the injection volume is 20 muL, the detection wavelength is 214nm, and the main chromatographic peak is collected.
By adopting the technical scheme, the spleen aminopeptide mainly comprises polypeptide, nucleotide, free amino acid and other components, the components are complex, and the molecular weights of the components are different. The method adopts a chromatographic separation technology, the chromatographic separation is an effective method for separating various components in a complex mixture, different substances have different distribution coefficients in a system formed by a fixed phase and a mobile phase, and when the mobile phase and the fixed phase move relatively, the substances move along with the mobile phase and are repeatedly distributed between the two phases, so that the components are separated.
In the chromatographic separation condition, the selection of a mobile phase is important for obtaining a proper peak pattern and successfully separating the polypeptide from the spleen aminopeptide, a gradient elution mode is selected, the mobile phase A is a formic acid aqueous solution, the mobile phase B comprises acetonitrile, formic acid and adipic acid, and the acetonitrile and formic acid are commonly used mobile phase components in the chromatographic separation.
The invention is further provided with: step two, sample treatment, namely taking the spleen aminopeptide stock solution for centrifugation, wherein the centrifugation conditions are as follows: centrifuging at 12000rpm for 10min at 4 deg.C, collecting supernatant, adding into initial mobile phase, and filtering with 0.22 μm organic membrane to obtain sample solution.
By adopting the technical scheme, when a sample is treated, the spleen aminopeptide stock solution is taken for centrifugation, the centrifugation is carried out for 10 minutes at 4 ℃, and then the centrifugation is carried out through an organic membrane with the aperture of 0.22 mu m, so that macromolecular impurities in the spleen aminopeptide stock solution are removed, the interference of macromolecular substances during chromatographic separation is reduced, and the accuracy of polypeptide extraction by chromatographic separation is improved.
The invention is further provided with: in step two, 100. mu.L of the starting mobile phase was added per 600. mu.L of supernatant.
By adopting the technical scheme, 100 mu L of initial mobile phase is added into every 600 mu L of supernatant, the initial mobile phase refers to the initial mobile phase when chromatographic separation gradient elution is carried out, and the initial mobile phase comprises 95% of mobile phase A and 5% of mobile phase B according to the chromatographic conditions in the step three.
The invention is further provided with: and step two, sample treatment, namely dissolving the spleen aminopeptide freeze-dried powder in deionized water, dialyzing by using a dialysis bag at 4 ℃, adding an initial mobile phase into the dialyzed spleen aminopeptide stock solution, and filtering by using an organic membrane of 0.22 mu m to obtain a sample solution.
By adopting the technical scheme, when the spleen aminopeptide is freeze-dried powder, the spleen aminopeptide freeze-dried powder is dissolved in deionized water, dialysis is carried out by using a dialysis bag, the molecular weight cut-off of the dialysis bag can be selected according to the size of target polypeptide, an initial mobile phase is added into the spleen aminopeptide stock solution after dialysis, and a sample solution is obtained after filtration by an organic membrane.
The invention is further provided with: in step two, 200. mu.L of the starting mobile phase was added per 500. mu.L of the spleen aminopeptide stock solution.
By adopting the technical scheme, 200 mu L of initial mobile phase is added into every 500 mu L of spleen aminopeptide stock solution.
The invention is further provided with: and step three, collecting chromatographic peaks of 11.222-11.711min, 25.719-25.919min, 25.935-26.285min, 26.302-26.652min, 30.016-30.240min and 30.999-31.049min in separation and purification.
By adopting the technical scheme, under the chromatographic condition of the step three, the main chromatographic peaks comprise the 6 time periods, the polypeptides in the spleen aminopeptide can be concentrated in the 6 chromatographic peaks, and the separated polypeptides can be obtained by collection.
In conclusion, the invention has the following beneficial effects: the polypeptide in the spleen aminopeptide is separated and extracted by adopting a chromatograph, and when a sample is treated, the sample is filtered by using an organic membrane with the diameter of 0.22 mu m, so that macromolecular substances in the spleen aminopeptide sample are removed, and the interference on the extraction of the polypeptide is reduced; secondly, after sample injection, gradient elution is carried out, and the polypeptide is eluted at the main chromatographic peak in a centralized manner by adjusting the proportion between the mobile phase A and the mobile phase B, so that the effects of extracting the polypeptide in the spleen aminopeptide and having good separation and purification effects are achieved.
Detailed Description
Example 1: a method for extracting polypeptide in spleen aminopeptide comprises the following steps of preparing a reagent, and preparing a mobile phase A and a mobile phase B, wherein a solvent in the mobile phase A is water, a solute is formic acid with the mass percent concentration of 0.1, a solvent in the mobile phase B is acetonitrile, and the solute comprises formic acid with the mass percent concentration of 0.1% and adipic acid with the mass percent concentration of 0.05%.
Step two, sample treatment, namely taking the spleen aminopeptide stock solution for centrifugation, wherein the centrifugation conditions are as follows: centrifuging at 12000rpm for 10min at 4 deg.C, collecting supernatant, adding into initial mobile phase, adding 100 μ L initial mobile phase into each 600 μ L supernatant, and filtering with 0.22 μm organic membrane to obtain sample solution. Wherein the starting mobile phase comprises 95% mobile phase a and 5% mobile phase B by volume.
And step three, separating and purifying, wherein the chromatographic conditions are adopted, the filler of the chromatographic column is porous silica gel, the diameter of the filler particles is 5 mu m, the inner diameter of the chromatographic column is 2.1mm, the length of the chromatographic column is 75mm, and the chromatographic column is Agilent Poroshell 300 SB-C18. The column temperature is 40 ℃, gradient elution is carried out for 0min-5min-14min-18min-24.5min-30min, and the mobile phase B is 5% -12% -17% -17.5% -30% -50%; the flow rate is 0.5mL/min, the sample injection volume is 20 muL, the detection wavelength is 214nm, and chromatographic peaks of 11.222-11.711min, 25.719-25.919min, 25.935-26.285min, 26.302-26.652min, 30.016-30.240min and 30.999-31.049min are collected.
Samples of each time period were collected, and the nucleotide content, sugar content, and polypeptide content in each sample were measured, and the measurement results are listed in table 2.
Example 2: a method for extracting polypeptide in spleen aminopeptide is different from the embodiment 1 in the step two, sample treatment is carried out, spleen aminopeptide freeze-dried powder is taken and dissolved in deionized water, dialysis is carried out by a dialysis bag at the temperature of 4 ℃, an initial mobile phase is added into a dialyzed spleen aminopeptide stock solution, 200 mu L of the initial mobile phase is added into every 500 mu L of the spleen aminopeptide stock solution, and the mixture is filtered by an organic membrane with the thickness of 0.22 mu m to obtain a 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 | Time period/min | Nucleotide (mu g/ml) | Candy (mug/ml) | Polypeptides (ug/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 | Is free of | 1.2-2 | 0.4-0.5 |
| Fraction5 | 25.935-26.285 | Is free of | 1.2-2 | 0.4-0.5 |
| Fraction6 | 26.302-26.652 | Is free of | 1.2-2 | 0.4-0.5 |
| Fraction7 | 26.652-30.016 | Is free of | 1.2-2 | 0.1-0.2 |
| Fraction8 | 30.016-30.240 | Is free of | 1.2-2 | 0.4-0.5 |
| Fraction9 | 30.999-31.049 | Is free of | 1.2-2 | 0.4-0.5 |
Comparative example: the difference between the method for extracting the polypeptide in the spleen aminopeptide and the embodiment 1 is that the solvent in the mobile phase B is acetonitrile, and the solute comprises 0.1 mass percent of formic acid.
Samples at each time period were collected, and the nucleotide content, sugar content, and polypeptide content in each sample were measured, and the measurement results are listed in table 3.
TABLE 3
| Sample name | Time period/min | Nucleotide (mu g/ml) | Candy (mug/ml) | Polypeptides (ug/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 | Is free of | 0.2-0.3 |
| Fraction8 | 30.016-30.240 | 4-5 | Is free of | 0.2-0.3 |
| Fraction9 | 30.999-31.049 | 4-5 | Is free of | 0.05-0.1 |
The specific embodiments are only for explaining the present invention, and the present invention is not limited thereto, and those skilled in the art can make modifications without inventive contribution to the present embodiments as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present invention.
Claims (6)
1. A method for extracting polypeptide in spleen aminopeptide is characterized in that: comprises the following steps
Preparing a reagent, namely preparing a mobile phase A and a mobile phase B, wherein a solvent in the mobile phase A is water, a solute is formic acid with the mass percent concentration of 0.1, a solvent in the mobile phase B is acetonitrile, and the solute comprises formic acid with the mass percent concentration of 0.1% and adipic acid with the mass percent concentration of 0.05%;
step two, sample treatment, namely adding spleen aminopeptide into the initial mobile phase to obtain a sample solution;
step three, separating and purifying, wherein the chromatographic conditions are adopted, Agilent Poroshell 300SB-C18 is selected as a chromatographic column, the filler of the chromatographic column is porous silica gel, the diameter of the filler particles is 5 mu m, the inner diameter of the chromatographic column is 2.1mm, the length of the chromatographic column is 75mm, the temperature of the chromatographic column is 40 ℃, gradient elution is carried out for 0min-5min-14min-18min-24.5min-30min, and a mobile phase B is 5% -12% -17% -17.5% -30% -50%; the flow rate is 0.5mL/min, the injection volume is 20 muL, the detection wavelength is 214nm, and the main chromatographic peak is collected.
2. The method for extracting polypeptide in spleen amino-peptide according to claim 1, wherein the method comprises the following steps: step two, sample treatment, namely taking the spleen aminopeptide stock solution for centrifugation, wherein the centrifugation conditions are as follows: centrifuging at 12000rpm for 10min at 4 deg.C, collecting supernatant, adding into initial mobile phase, and filtering with 0.22 μm organic membrane to obtain sample solution.
3. The method for extracting polypeptide in spleen amino-peptide according to claim 2, wherein the method comprises the following steps: in step two, every 600. mu.L of supernatant was added to 100. mu.L of the starting mobile phase.
4. The method for extracting polypeptide in spleen amino-peptide according to claim 1, wherein the method comprises the following steps: and step two, sample treatment, namely dissolving the spleen aminopeptide freeze-dried powder in deionized water, dialyzing by using a dialysis bag at 4 ℃, adding an initial mobile phase into the dialyzed spleen aminopeptide stock solution, and filtering by using an organic membrane of 0.22 mu m to obtain a sample solution.
5. The method for extracting polypeptide in spleen amino-peptide according to claim 4, wherein the method comprises the following steps: in step two, 200. mu.L of the starting mobile phase was added per 500. mu.L of the spleen aminopeptide stock solution.
6. The method for extracting polypeptide in spleen amino-peptide according to claim 1, wherein the method comprises the following steps: and step three, collecting chromatographic peaks of 11.222-11.711min, 25.719-25.919min, 25.935-26.285min, 26.302-26.652min, 30.016-30.240min and 30.999-31.049min in separation and purification.
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| 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 |
| CN114940698B (en) * | 2022-07-22 | 2022-10-21 | 北京第一生物化学药业有限公司 | Method for extracting acidic polypeptide from spleen aminopeptide stock solution |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN102167726A (en) * | 2011-02-21 | 2011-08-31 | 山东省莘县金鑫畜禽有限公司 | Method for separating and extracting active polypeptide and immune ribonucleic acid by utilizing blood and spleen of animal |
| CN107298706A (en) * | 2017-07-31 | 2017-10-27 | 河南科技学院 | A kind of holstein cow spleen derived antimicrobial peptide and preparation method and application |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1298736C (en) * | 2005-04-21 | 2007-02-07 | 石家庄三鹿集团股份有限公司 | Method for separating purified immuno regulation active polypeptide from placenta of cattle |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN102167726A (en) * | 2011-02-21 | 2011-08-31 | 山东省莘县金鑫畜禽有限公司 | Method for separating and extracting active polypeptide and immune ribonucleic acid by utilizing blood and spleen of animal |
| CN107298706A (en) * | 2017-07-31 | 2017-10-27 | 河南科技学院 | A kind of holstein cow spleen derived antimicrobial peptide and preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| 兽用鸡脾转移因子的多肽组成研究;黄迪海等;《中国兽药杂志》;20141231;第48卷(第2期);第23-27页 * |
| 山羊脾脏多肽的分离纯化;杨兆勇等;《药物分析杂志》;20051231;第25卷(第8期);第962-963页 * |
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