CN108129549B - Method for extracting polypeptide in spleen aminopeptide - Google Patents
Method for extracting polypeptide in spleen aminopeptide Download PDFInfo
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- CN108129549B CN108129549B CN201711377944.0A CN201711377944A CN108129549B CN 108129549 B CN108129549 B CN 108129549B CN 201711377944 A CN201711377944 A CN 201711377944A CN 108129549 B CN108129549 B CN 108129549B
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- mobile phase
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- polypeptide
- spleen aminopeptide
- 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
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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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
-
- 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 method for extracting polypeptide in spleen aminopeptide, which comprises the following steps of firstly, 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; and step three, separating and purifying. The invention has the following advantages and effects: adopting a chromatographic separation technology, adsorbing substances such as polypeptide, sugar, free amino acid, nucleotide and the like in the spleen aminopeptide by a chromatographic column, and eluting the polypeptide from the chromatographic column in a concentrated manner in the gradient elution process; in the chromatographic separation process, the proper mobile phase and chromatographic column are selected, the polypeptide in the spleen aminopeptide can be intensively eluted, the intervals among a plurality of chromatographic peaks are large, and the effects of separating and purifying the polypeptide in the spleen aminopeptide, good separation effect and simple operation 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, 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 the formic acid with the mass percent concentration of 0.1% and cyclohexyl ether with the mass percent concentration of 0.1%; step two, sample treatment, namely adding spleen aminopeptide into the initial mobile phase to obtain a sample solution; step three, separation and purification, and chromatographic conditions: the chromatographic column is Agilent Poroshell300SB-C18, the filler particles in the chromatographic column are 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-13min-13.5min-26min-35min, and a mobile phase B5% -11.2% -15.8% -36% -60%; the flow rate is 0.5ml/min, the sample injection volume is 20 muL, the detection wavelength is 214nm, and chromatographic peaks with peak time of 16.060-16.462min, 23.072-24.022min and 29.968-31.451min are collected to obtain the separated polypeptide.
By adopting the technical scheme, 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 stationary phase and a mobile phase, and when the mobile phase and the stationary 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.
The main components in the spleen aminopeptide comprise polypeptide, free amino acid, nucleotide and sugar, the application adopts a chromatographic separation technology to separate and purify the polypeptide from the spleen aminopeptide, in the chromatographic separation condition, a silica gel column is selected as a chromatographic column, a solvent in a mobile phase A is water, a solute is formic acid, a solvent in a mobile phase B is acetonitrile, and solutes are formic acid and cyclohexyl ether, a gradient elution method is adopted, and when 0-13min is carried out, the mobile phase component comprises 95% of the mobile phase A and 5% of the mobile phase B, and the free amino acid and the nucleotide in the spleen aminopeptide are eluted from the chromatographic column; 13-13.5min, wherein the mobile phase component comprises 88.8% of mobile phase A and 11.2% of mobile phase B, and the nucleotides and sugar in the spleen aminopeptide are eluted from the chromatographic column. The components are separated from each other in sequence by a gradient elution mode, and due to the complex components in the spleen aminopeptide, when the polypeptide in the spleen aminopeptide is separated and purified by a chromatographic separation technology, the polypeptide is difficult to be thoroughly separated from other components in the spleen aminopeptide, such as sugar, nucleotide and the like. Therefore, in the application, the cyclohexadene is added into the mobile phase B, the peak appearance trend can be obviously improved in the gradient elution process, under the chromatographic condition of the step three, the polypeptides in the spleen aminopeptide are concentrated at chromatographic peaks with the peak appearance time of 16.060-16.462min, 23.072-24.022min and 29.968-31.451min, the polypeptides in the spleen aminopeptide can be separated from the spleen aminopeptide, and the effect of separating and purifying the polypeptides in the spleen aminopeptide is achieved.
The invention is further provided with: and step four, freeze drying, namely performing freeze drying treatment on the sample obtained in the step three to obtain freeze-dried powder.
By adopting the technical scheme, the freeze drying is vacuum freeze drying, freeze drying for short, which is a drying technology for freezing wet materials or solution into a solid state at a lower temperature, then directly sublimating water in the solid state into a gaseous state without liquid state under vacuum, and finally dehydrating the materials.
The invention is further provided with: and step two, sample treatment, namely dialyzing the spleen aminopeptide stock solution by using a dialysis bag, adding an initial mobile phase into the dialyzed spleen aminopeptide stock solution, and filtering by using a 0.22-micron organic membrane to obtain a sample solution.
By adopting the technical scheme, in the sample treatment, the spleen aminopeptide stock solution is dialyzed firstly, the initial mobile phase is added after the dialysis treatment, in the chromatographic condition of the third step, the components of the initial mobile phase are 95% of mobile phase A and 5% of mobile phase B, small molecular substances in the spleen aminopeptide stock solution are removed through dialysis, and then impurities such as macromolecular proteins are removed through filtration.
The invention is further provided with: step two, in the sample treatment, 200 mu L of initial mobile phase is added into every 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: 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.
In conclusion, the invention has the following beneficial effects: the chromatographic separation technology is adopted, the chromatographic column adsorbs substances such as polypeptide, sugar, free amino acid, nucleotide and the like in the spleen aminopeptide, and the polypeptide is intensively eluted from the chromatographic column in the gradient elution process, so that the effects of separating and purifying the polypeptide in the spleen aminopeptide and having good separation effect 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 the formic acid with the mass percent concentration of 0.1% and cyclohexyl ether with the mass percent concentration of 0.1%.
And step two, sample treatment, namely dialyzing the spleen aminopeptide stock solution by using a dialysis bag, adding an initial mobile phase into the dialyzed spleen aminopeptide stock solution, adding 200 mu L of initial mobile phase into every 500 mu L of the spleen aminopeptide stock solution, and filtering by using a 0.22 mu m organic membrane to obtain a sample solution.
Step three, separation and purification, and chromatographic conditions: the chromatographic column is a silica gel column, the filler particles are 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 chromatographic column is Agilent Poroshell300SB-C18, the column temperature is 40 ℃, gradient elution is carried out for 0min-13min-13.5min-26min-35min, and the mobile phase B is 5% -11.2% -15.8% -36% -60%, as shown in Table 1; the flow rate was 0.5ml/min, the injection volume was 20. mu.L, the detection wavelength was 214nm, and the samples were collected in stages for the time periods shown in Table 2.
Identification test: and (3) determining the total sugar content, the nucleotide content and the polypeptide content in the sample in each time period, wherein the polypeptide content is detected by using a BCA kit, and the detection results are listed in Table 2.
Collecting chromatographic peaks with peak time of 16.060-16.462min, 23.072-24.022min and 29.968-31.451min to obtain separated polypeptide.
And step four, freeze drying, namely performing freeze drying treatment on the sample obtained in the step three to obtain freeze-dried powder.
TABLE 1
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 95 | 5 |
| 13 | 95 | 5 |
| 13.5 | 95 | 5 |
| 26 | 95 | 5 |
| 35 | 95 | 5 |
TABLE 2
| Sample name | Time period/min | Nucleotide (mu g/ml) | Candy (mug/ml) | Polypeptides (ug/ml) |
| Fraction1 | 0-16.060 | ＞20 | 2.5-5 | 0.025-0.05 |
| Fraction2 | 16.060-16.462 | 8-12 | 2.5-5 | 0.4-0.5 |
| Fraction3 | 16.462-23.072 | 8-12 | 2.5-5 | 0.1-0.2 |
| Fraction4 | 23.072-24.022 | Is free of | 2.5-5 | 0.4-0.5 |
| Fraction5 | 24.022-29.968 | Is free of | 2.5-5 | 0.1-0.2 |
| Fraction6 | 29.968-31.451 | Is free of | 2.5-5 | 0.4-0.5 |
| Fraction7 | 31.451-35 | Is free of | ＞60 | 0.1-0.2 |
As can be seen from the test results in Table 2, the peak-appearing time of the polypeptide in the spleen aminopeptide is concentrated in 16.060-16.462min, 23.072-24.022min and 29.968-31.451 min.
Example 2: the difference between the method for extracting the polypeptide in the spleen aminopeptide and the example 1 is that in the second step, a sample is processed, and a spleen aminopeptide stock solution is taken 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.
Comparative example: a method for extracting polypeptide in spleen aminopeptide is different from the method in example 1 in that acetonitrile is used as a solvent in a mobile phase B, formic acid with the solute concentration of 0.1% by mass is used as a solute, samples in all time periods are collected and subjected to identification tests, and the test results are listed in Table 3.
TABLE 3
| Sample name | Time period/min | Nucleotide (mu g/ml) | Candy (mug/ml) | Polypeptides (ug/ml) |
| Fraction1 | 0-16.060 | 15-20 | 2.5-5 | 0.025-0.05 |
| Fraction2 | 16.060-16.462 | 12-17 | 2.5-5 | 0.3-0.4 |
| Fraction3 | 16.462-23.072 | 8-12 | 2.5-5 | 0.3-0.4 |
| Fraction4 | 23.072-24.022 | 5-8 | 2.5-5 | 0.3-0.4 |
| Fraction5 | 24.022-29.968 | 5-8 | 2.5-5 | 0.3-0.4 |
| Fraction6 | 29.968-31.451 | Is free of | 2.5-5 | 0.2-0.3 |
| Fraction7 | 31.451-35 | Is free of | ＞60 | 0.1-0.2 |
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. The method for extracting polypeptide in spleen aminopeptide is characterized by comprising 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 the formic acid with the mass percent concentration of 0.1% and cyclohexyl ether with the mass percent concentration of 0.1%;
step two, sample treatment, namely adding spleen aminopeptide into the initial mobile phase to obtain a sample solution;
step three, separation and purification, and chromatographic conditions: the chromatographic column is Agilent Poroshell300SB-C18, the filler particles in the chromatographic column are 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-13min-13.5min-26min-35min, and the mobile phase B is 5% -11.2% -15.8% -36% -60%; the flow rate is 0.5ml/min, the sample injection volume is 20 muL, the detection wavelength is 214nm, and chromatographic peaks with peak time of 16.060-16.462min, 23.072-24.022min and 29.968-31.451min are collected to obtain the separated polypeptide.
2. The method for extracting polypeptide in spleen amino-peptide according to claim 1, wherein the method comprises the following steps: and step four, freeze drying, namely performing freeze drying treatment on the polypeptide obtained in the step three to obtain freeze-dried powder.
3. 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 dialyzing the spleen aminopeptide stock solution by using a dialysis bag, adding an initial mobile phase into the dialyzed spleen aminopeptide stock solution, and filtering by using a 0.22-micron organic membrane to obtain a sample solution.
4. The method for extracting polypeptide in spleen amino-peptide according to claim 3, wherein the method comprises the following steps: step two, in the sample treatment, 200 mu L of initial mobile phase is added into every 500 mu L of the spleen aminopeptide stock solution.
5. 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.
6. The method for extracting polypeptide in spleen amino-peptide according to claim 5, 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.
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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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| WO2013089550A1 (en) * | 2011-12-16 | 2013-06-20 | Instituto Politecnico Nacional | Method for obtaining a dialyzable leukocyte extract |
| 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 |
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2017
- 2017-12-19 CN CN201711377944.0A patent/CN108129549B/en active Active
Patent Citations (5)
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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 |
| 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 |
| WO2013089550A1 (en) * | 2011-12-16 | 2013-06-20 | Instituto Politecnico Nacional | Method for obtaining a dialyzable leukocyte extract |
| 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 |
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| Title |
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| 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 * |
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