Preparation method of protamine sulfate

Abstract

The invention discloses a preparation method of protamine sulfate, and belongs to the field of biological medicine. The preparation method of the protamine sulfate comprises the following steps: S1, crushing mature testis tissues of aquatic organisms, adding saline water, stirring I and filtering I to obtain a salt extracting solution; s2, adding water and polyethyleneimine into the salt extracting solution, stirring II, and filtering II to obtain filtrate; s3, adding water and polyethyleneimine into the filtrate, stirring III, standing I, and pouring supernate to obtain an oily substance at the lower layer; s4, an ammonium sulfate solution is added into the oily matter, stirring IIII and standing II are conducted, supernate is poured out, oily matter at the bottom is obtained and dried, and protamine sulfate is obtained. The preparation method has the advantages of no use of inorganic acid, high purity, small pollution, reagent saving and low cost, and the obtained protein has good activity.

Description

Technical Field

[0001] The present application relates to a method for preparing protamine sulfate, and belongs to the field of biomedicine. Background Art

[0002] Protamine is a basic protein that binds to DNA and exists in the nucleus of mature sperm of fish such as herring and salmon and vertebrates such as chicken. Its relative molecular mass is about 6000-10000 and it is composed of about 30 amino acids, of which 2 / 3 are arginine. Protamine has important applications in medicine. For example, protamine sulfate is the only drug that can counteract heparin in extracorporeal heart surgery and can offset the anticoagulant effect of heparin or synthetic anticoagulants. Protamine also has anti-tumor, fatigue relief, liver function enhancement, and stimulating the pituitary gland to release gonadotropin. It also has a certain effect on the treatment of male infertility. In addition, protamine also has good antibacterial properties and can be used as a natural food preservative in the preservation of various foods.

[0003] In the prior art, acid-soluble cationic polymer flocculants are added to the inorganic acid extract to separate and remove impurity proteins, such as shell polyester, polymethacrylate, polyvinylamine, halogenated polydienyl ammonium, etc. However, these polymer flocculants have low selectivity for proteins and the extraction effect is not ideal. Summary of the invention

[0004] The present application provides a method for producing protamine with high purity using mature testicular tissue (milt) of aquatic organisms as raw material. Since most impurity proteins and nucleic acids are acidic, the selectivity of polyethyleneimine (PEI) can be used to highly selectively separate and purify protamine sulfate. It will not mix with impurities, and can simply and selectively extract protamine from milt, changing the situation where the inorganic acid extract contains impurity proteins, blood, mucus components, etc.

[0005] This application solves the above technical problems through the following technical solutions:

[0006] A method for preparing protamine sulfate comprises the following steps:

[0007] S1, crushing the mature testicular tissue of the aquatic organism, adding salt water, stirring for 1, filtering for 1, and obtaining a salt extract;

[0008] S2, adding water and polyethyleneimine to the salt extract, stirring II, filtering II, and obtaining a filtrate;

[0009] S3, add water and polyethyleneimine to the filtrate, stir III, let stand I, pour out the supernatant to obtain the oily substance in the lower layer;

[0010] S4, adding ammonium sulfate solution to the oily substance, stirring IIII, standing II, pouring out the supernatant to obtain the oily substance at the bottom, drying to obtain the protamine sulfate.

[0011] In the present application, the mature testicular tissue of aquatic organisms is also referred to as milt, specifically the milt of salmon, etc.

[0012] Optionally, in step S1, the brine is a sodium chloride aqueous solution.

[0013] Optionally, the concentration of the brine is 1 mol / L to 3 mol / L;

[0014] The amount of salt water added is 1.5 to 5 times the weight of the mature testicular tissue of the aquatic organism.

[0015] Optionally, the concentration of the brine is 1.5 mol / L to 2.5 mol / L;

[0016] The amount of salt water added is 2 to 4 times the weight of the mature testicular tissue of the aquatic organism.

[0017] Optionally, in step S1, stirring I is performed at 20 to 30° C. for 0.5 to 3.5 hours.

[0018] Optionally, in step S2 and step S3, the molecular weight of the polyethyleneimine is independently selected from 1000 to 25000.

[0019] Optionally, in step S2 and step S3, the molecular weight of the polyethyleneimine is independently selected from 1500 to 3000.

[0020] Optionally, in step S2, the mass concentration of polyethyleneimine in the final solution is 0.2-1%.

[0021] Wherein, in step S2, the mass concentration of polyethyleneimine in the final solution refers to the final solution concentration of polyethyleneimine after adding water.

[0022] Optionally, in step S2, the mass concentration of polyethyleneimine in the final solution is 0.5-0.98%.

[0023] Optionally, in step S2, the amount of water added is to adjust the final concentration of the brine to 1-2 mol / L.

[0024] Wherein, in step S2, the final concentration of the saline solution refers to the final concentration of the saline solution after adding water.

[0025] Optionally, in step S2, the amount of water added is to adjust the final concentration of the brine to 1.5-1.8 mol / L.

[0026] Optionally, in step S2, stirring II is performed at 50-80° C. for 0.5-1.5 hours.

[0027] Optionally, in step S3, the mass concentration of polyethyleneimine in the final solution is 0.2 to 0.96%.

[0028] Wherein, in step S3, the mass concentration of polyethyleneimine in the final solution refers to the final solution concentration of polyethyleneimine after adding water. Optionally, in step S3, the amount of water added is to adjust the final concentration of the brine to 0.1-1 mol / L.

[0029] Wherein, in step S3, the final concentration of the saline refers to the final concentration of the saline solution after adding water. And the final concentration of the saline in step S3 is less than the final concentration of the saline in step S2. Optionally, in step S3, the amount of water added is to adjust the final concentration of the saline to 0.5-1 mol / L.

[0030] Polyethyleneimine is a chain product formed by polymerization of ethyleneimine. It carries a positive charge in a neutral solution, so it can form an electrically neutral complex with acidic macromolecules (such as DNA, RNA, and acidic proteins), and the precipitates are cross-linked to produce floccules. The amount of protein precipitated by PEI is determined by the ionic strength of the solution. Its binding ability with proteins is salt-dependent. Under low ionic strength: PEI precipitates with acidic proteins; under higher ionic strength: PEI reacts with highly electronegative proteins to precipitate; under high ionic strength: all proteins are not precipitated. In addition, when the NaCl concentration in the solution is as high as 1 to 2M, PEI can still bind to DNA / RNA. Therefore, in the present application, in step S2, water and polyethyleneimine are added to the salt extraction liquid, and the amount of water added is controlled to adjust the final concentration of the salt water to 1 to 2 mol / L to precipitate nucleic acids in milt; in step S3, water and polyethyleneimine are added, and the amount of water added is controlled to adjust the final concentration of the salt water to 0.1 to 1 mol / L to precipitate protamine.

[0031] Optionally, in step S3, stirring III is performed at 20-30° C. for 0.5-1.5 hours.

[0032] Optionally, in step S4, the mass concentration of the ammonium sulfate solution is 20-30%;

[0033] The amount of ammonium sulfate solution added is 1.5 to 2.5 times the weight of the oil.

[0034] Optionally, in step S4, stirring IIII is performed at 50-80° C. for 0.5-2 hours.

[0035] As a specific embodiment, the preparation method of protamine sulfate comprises the following steps:

[0036] S1. Salt extraction: put mature testicular tissue (milt) of aquatic organisms into a beaker, break it, add 1.5 mol / L to 2 mol / L salt water, stir at room temperature for 0.5 to 3 hours, filter, and wash with water;

[0037] S2. Precipitate nucleic acid: add the salt extract into a beaker, heat to 50°C, add water and polyethyleneimine (PEI), stir, filter, and obtain a filtrate;

[0038] S3, PEI-protamine precipitation: add purified water and polyethyleneimine (PEI) to the supernatant, stir, let stand and pour to obtain an oily substance;

[0039] S4. Sulfate-protamine precipitation: add water to the oil, start stirring, then add ammonium sulfate, stir and heat to 50-80℃ until it dissolves, stir for 1 hour, stop stirring, let stand for 12 hours, discard the supernatant, get the oil at the bottom, and vacuum dry; protamine sulfate is obtained.

[0040] The beneficial effects of this application include:

[0041] 1) The preparation method of protamine sulfate provided in the present application uses high-concentration salt water for extraction during the salt extraction process to avoid the use of inorganic acids. It not only reduces environmental pollution, but also improves the shortcomings of traditional methods: the inorganic acid extract contains impurity proteins such as histones and components such as blood and mucus. After obtaining the high-purity salt extract, the protamine sulfate can be directly separated and purified using PEI with high selectivity to further purify the protamine sulfate. Protamine sulfate is directly purified during the preparation process, avoiding repeated dissolution and extraction of multiple refinements, saving reagents, and low cost.

[0042] 2) Compared with processes such as enzymatic hydrolysis, chromatography, dialysis and ultrafiltration, which require special equipment, the method for preparing protamine sulfate provided in the present application is simpler and more practical, does not rely on specific labels, does not require large-scale instruments such as protein purification systems, is low-cost, and the obtained protein has good activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the absorption spectrum of the protamine sulfate prepared in Example 1;

[0044] Figure 2 This is the absorption spectrum of protamine sulfate prepared in Comparative Example 1;

[0045] Figure 3 This is the absorption spectrum of protamine sulfate prepared in Comparative Example 4;

[0046] Figure 4 This is the absorption spectrum of the protamine sulfate prepared in Example 3;

[0047] Figure 5 This is the liquid chromatogram of the protamine sulfate prepared in Example 3. DETAILED DESCRIPTION

[0048] The present application is described in detail below with reference to embodiments, but the present application is not limited to these embodiments.

[0049] Unless otherwise specified, the raw materials in the examples of this application were purchased through commercial channels.

[0050] Unless otherwise specified, conventional methods were used for testing and instrument settings were those recommended by the manufacturer.

[0051] Polyethyleneimine was purchased from MacLean's reagent, a 50% aqueous solution, with a molecular weight of 2000. In the examples, the polyethyleneimine (PEI) aqueous solution refers to a 50% polyethyleneimine aqueous solution.

[0052] Example 1

[0053] A method for preparing protamine sulfate comprises the following steps:

[0054] (1) Salt extraction: accurately weigh 1000 g of salmon milt, add 2000 g of 2 mol / L sodium chloride aqueous solution, stir at room temperature for 1 hour, filter, and obtain a salt extract;

[0055] (2) Precipitating nucleic acids: Add the salt extract to a beaker, add 40 g of polyethyleneimine (PEI) aqueous solution and 600 g of purified water, the mass concentration of polyethyleneimine in the final solution is 0.76%, the final concentration of the brine is 1.53 mol / L, stir at 50°C for 1 hour, filter, and obtain a filtrate.

[0056] (3) PEI-protamine precipitation: Add 2000 g of purified water and 50 g of polyethyleneimine (PEI) aqueous solution to the filtrate. In the final solution, the mass concentration of polyethyleneimine is 0.74% and the final concentration of brine is 0.85 mol / L. Stir at room temperature for 1 hour, let stand for 12 hours, and pour out the lower layer of oil;

[0057] (4) Sulfate-protamine precipitation: 100 g of 20% ammonium sulfate solution was added to 52.49 g of the oily substance, the temperature was raised to 50° C., and the mixture was stirred to dissolve. The mixture was stirred for one hour, and the mixture was allowed to stand at room temperature for 12 hours to precipitate. The supernatant was removed, and the precipitate was vacuum dried and weighed to obtain 34.82 g of protamine sulfate.

[0058] Example 2

[0059] A method for preparing protamine sulfate comprises the following steps:

[0060] (1) Salt extraction: accurately weigh 500 g of blue salmon milt, add 2000 g of 2 mol / L sodium chloride aqueous solution, stir at room temperature for 3 hours, filter, and obtain a salt extract;

[0061] (2) Precipitating nucleic acids: Add the salt extract to a beaker, add 40 g of polyethyleneimine (PEI) aqueous solution and 500 g of purified water, the mass concentration of polyethyleneimine in the final solution is 0.98%, the final concentration of the brine is 1.57 mol / L, stir at 50°C for 0.5 hour, filter, and obtain a filtrate.

[0062] (3) PEI-protamine precipitation: Add 2000 g of purified water and 20 g of polyethyleneimine (PEI) aqueous solution to the supernatant. In the final solution, the mass concentration of polyethyleneimine is 0.44% and the final concentration of saline is 0.88 mol / L. Stir at room temperature for 1 hour, let stand for 12 hours, and pour out the lower layer of oil;

[0063] (4) Sulfate-protamine precipitation: 50 g of 25% ammonium sulfate solution was added to 22.46 g of the oily substance, the temperature was raised to 60°C, and the mixture was stirred to dissolve. The mixture was stirred for one hour, and allowed to stand at room temperature for 12 hours to precipitate. The supernatant was removed, and the precipitate was vacuum dried and weighed to obtain 16.71 g of protamine sulfate.

[0064] Example 3

[0065] A method for preparing protamine sulfate comprises the following steps:

[0066] (1) Salt extraction: accurately weigh 1000 g of salmon milt, add 2000 g of 1.5 mol / L sodium chloride aqueous solution, stir at room temperature for 2 hours, and filter to obtain a salt extract.

[0067] (2) Precipitating nucleic acids: Add the salt extract to a beaker and add 30 g of polyethyleneimine (PEI) aqueous solution. In the final solution, the mass concentration of polyethyleneimine is 0.74% and the final concentration of saline is 1.5 mol / L. Stir at 50° C. for 1 hour and filter to obtain a filtrate.

[0068] (3) PEI-protamine precipitation: Add 2000 g of purified water and 30 g of polyethyleneimine (PEI) aqueous solution to the supernatant, so that the mass concentration of polyethyleneimine in the final solution is 0.56% and the final concentration of saline is 0.74 mol / L. Stir at room temperature for 30 minutes, let stand for 12 hours, and pour out to obtain the lower layer of oil.

[0069] (4) Sulfate-protamine precipitation: 100 g of ammonium sulfate solution with a mass concentration of 28% was added to 45.82 g of the oily substance, the temperature was raised to 80° C., and the mixture was stirred to dissolve. The mixture was stirred for one hour, and the mixture was allowed to stand at room temperature for 12 hours to precipitate. The supernatant was removed, and the precipitate was vacuum dried and weighed to obtain 32.89 g of protamine sulfate.

[0070] Example 4

[0071] A method for preparing protamine sulfate comprises the following steps:

[0072] (1) Salt extraction: accurately weigh 500 g of salmon milt, add 2000 g of 2.3 mol / L sodium chloride aqueous solution, stir at room temperature for 2 hours, filter and obtain a salt extract;

[0073] (2) Precipitating nucleic acids: Add the salt extract to a beaker, add 30 g of polyethyleneimine (PEI) aqueous solution and 600 g of purified water, the mass concentration of polyethyleneimine in the final solution is 0.57%, the final concentration of the brine is 1.77 mol / L, stir at 52°C for 1 hour, filter, and obtain a filtrate.

[0074] (3) PEI-protamine precipitation: Add 2000 g of purified water and 30 g of polyethyleneimine (PEI) aqueous solution to the supernatant. In the final solution, the mass concentration of polyethyleneimine is 0.48%, and the final concentration of saline is 0.99 mol / L. Stir at room temperature for 30 minutes, let stand for 12 hours, and pour out the lower layer of oil;

[0075] (4) Sulfate-protamine precipitation: add 15 g of 22% ammonium sulfate solution to 7.5 g of the oily substance, heat to 80°C, stir to dissolve, continue stirring for one hour, let stand at room temperature for 12 hours, precipitate, remove the supernatant, vacuum dry the precipitate, weigh, and obtain 5.4 g of protamine sulfate.

[0076] Comparative Example 1

[0077] A method for preparing protamine sulfate comprises the following steps:

[0078] (1) Acid extraction: accurately weigh 1000 g of blue salmon milt, add 2000 g of 0.5 mol / L sulfuric acid solution and 10 g of diatomaceous earth, stir at 25° C. for 1 hour, and filter to obtain a filtrate;

[0079] (2) adding 30 wt % sodium hydroxide solution to the filtrate to adjust the pH to 7;

[0080] (3) Add 50 g of activated carbon to the filtrate, raise the temperature to 70°C, stir for one hour, and filter hot to obtain a filtrate;

[0081] (4) Add 3000 g of ethanol to the filtrate, let it stand at 0°C for 15 hours to precipitate, remove the supernatant, vacuum dry the precipitate, and weigh it to obtain 38.54 g of protamine sulfate.

[0082] Comparative Example 2

[0083] A method for preparing protamine sulfate comprises the following steps:

[0084] (1) Acid extraction: accurately weigh 1000 g of blue salmon milt, add 2000 g of 0.5 mol / L sulfuric acid solution and 10 g of diatomaceous earth, stir at 25° C. for 1 hour, and filter to obtain a filtrate;

[0085] (2) adding 30 wt % sodium hydroxide solution to the filtrate to adjust the pH to 7;

[0086] (3) adding 2.5 kg of chitosan to the filtrate, stirring at room temperature for one hour, letting it stand and pouring it to obtain a filtrate;

[0087] (4) Add 3000 g of ethanol to the filtrate, let it stand at 0°C for 15 hours to precipitate, remove the supernatant, vacuum dry the precipitate, and weigh it to obtain 36.77 g of protamine sulfate.

[0088] Comparative Example 3

[0089] A method for preparing protamine sulfate comprises the following steps:

[0090] (1) Salt extraction: accurately weigh 1000 g of blue salmon milt, add 2000 g of 2 mol / L sodium chloride solution and 10 g of diatomaceous earth, stir at 25° C. for 1 hour, and filter to obtain a filtrate;

[0091] (2) adding 2.5 kg of chitosan to the filtrate, stirring at room temperature for one hour, letting it stand and pouring it to obtain a supernatant;

[0092] (3) Add ammonium sulfate to the supernatant, raise the temperature to 50°C, stir to dissolve, continue stirring for one hour, let stand at room temperature for 12 hours, precipitate, remove the supernatant, vacuum dry the precipitate, and weigh to obtain 36.43 g of protamine sulfate.

[0093] Comparative Example 4

[0094] A method for preparing protamine sulfate comprises the following steps:

[0095] (1) Salt extraction: accurately weigh 1000 g of blue salmon milt, add 2000 g of 2 mol / L sodium chloride solution, stir at 25° C. for 1 hour, and filter to obtain a filtrate;

[0096] (2) adding 500 g of polyethyleneimine (PEI) aqueous solution to the filtrate, stirring at 50° C. for one hour, and filtering to obtain a filtrate;

[0097] (3) Add ammonium sulfate to the supernatant, raise the temperature to 50°C, stir to dissolve, continue stirring for one hour, let stand at room temperature for 12 hours, precipitate, remove the supernatant, vacuum dry the precipitate, and weigh to obtain 36.00 g of protamine sulfate.

[0098] Determination of absorbance of protamine sulfate

[0099] Take 0.2g of the protamine sulfate product prepared in the above Examples 1-4 and Comparative Examples 1-4, respectively, dissolve it in water and dilute it to 10.0ml. Take 2.5ml of the solution, dilute it to 5ml with water, and scan its absorption spectrum. The testing instrument is a UV-visible spectrophotometer. Among them, Figure 1 This is the absorption spectrum of the protamine sulfate prepared in Example 1; Figure 2 This is the absorption spectrum of protamine sulfate prepared in Comparative Example 1; Figure 3 This is the absorption spectrum of protamine sulfate prepared in Comparative Example 4;

[0100] Figure 4 This is the absorption spectrum of the protamine sulfate prepared in Example 3. The absorbances corresponding to 260nm and 280nm are shown in Table 2.

[0101] Identification of Protamine Sulfate Content

[0102] The content of protamine sulfate was determined by high performance liquid chromatography, and the specific chromatographic conditions were as follows:

[0103] Chromatographic column: End-capped solid octadecylsilane bonded silica gel as filler; 4.0×150mm; 3.6μm (Fenomi AerlsTM 3.6μm WIDEPOREX B-C18)

[0104] Column temperature: 50°C

[0105] Injection volume: 30 μl

[0106] Flow rate: 1.5ml / min

[0107] Wavelength: 210nm

[0108] Solvent: 618g / L hydrochloric acid solution; 1.03g / L hydrochloric acid solution (0.4:99.6)

[0109] Test solution: Take 30 mg of the test sample and place it in a 100 ml volumetric flask, add 400 μl of 618 g / L hydrochloric acid solution to dissolve it, dilute to the scale with 1.03 g / L hydrochloric acid solution, and shake well.

[0110] Reference solution a: Take 1 vial of protamine sulfate USP reference, dissolve it with 40 μl 618 g / L hydrochloric acid solution, dilute it with 1.03 g / L hydrochloric acid solution and make up to 10 ml, shake well.

[0111] Reference solution b: Accurately measure 1 ml of reference solution a and place it in a 50 ml volumetric flask, dilute to the mark with 1.03 g / L hydrochloric acid solution, and shake well.

[0112] Mobile phase: trifluoroacetic acid-acetonitrile-water (0.1:5:95) was used as mobile phase A, and trifluoroacetic acid-acetonitrile-water (0.1:52:48) was used as mobile phase B.

[0113] The gradient elution conditions are shown in Table 1:

[0114] Table 1

[0115]

[0116] Determination method: Accurately measure 30 μl of solvent, reference solution and test solution respectively, inject them into high performance liquid chromatography, and record the chromatogram.

[0117] Calculation method: Calculated by peak area according to external standard method

[0118]

[0119]

[0120] F: absolute correction factor;

[0122] : The average value of the absolute correction factors of the two reference solutions;

[0123] C 对 : Concentration of reference solution;

[0124] A 样 : The sum of the peak areas of all chromatographic peaks in the test solution with RRT greater than the reporting limit in the range of 0.80-1.30, taking protamine peptide A as the reference;

[0125] A 对 : The sum of the peak areas of all chromatographic peaks with RRT greater than the reporting limit in the range of 0.80-1.30 in the reference solution with protamine peptide A as the reference;

[0126] Vs 样 : dilution multiple of the test solution;

[0127] m 样 : Sample quantity of test product (mg);

[0128] D: Loss on drying data, % (according to the 2020 edition of the Chinese Pharmacopoeia 0831 loss on drying method);

[0129] S: Sulfate data, % (according to the sulfate determination method in 0802 of the 2020 edition of the Chinese Pharmacopoeia).

[0130] The protamine sulfate products prepared in the above Examples 1-4 and Comparative Examples 1-4 were identified according to the above method. Taking Example 3 as a typical example, its liquid chromatogram is as follows Figure 5 shown.

[0131] The statistical results are shown in Table 2 below:

[0132] Table 2

[0133]

[0134] As can be seen from the above table, the method of preparing protamine sulfate using polyethyleneimine (PEI) in the present application has higher selectivity than other adsorbents, and the prepared protamine sulfate has a higher content and less mixed nucleic acids and impurities.

[0135] The above are only a few embodiments of the present application and do not constitute any form of limitation to the present application. Although the present application is disclosed as above with preferred embodiments, it is not intended to limit the present application. Any technician familiar with the profession, without departing from the scope of the technical solution of the present application, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. A method for preparing protamine sulfate, characterized in that: The following steps are involved: S1, crushing the mature testicular tissue of the aquatic organism, adding salt water, stirring for 1, filtering for 1, and obtaining a salt extract; S2, adding water and polyethyleneimine to the salt extract, stirring II, filtering II, and obtaining a filtrate; S3, add water and polyethyleneimine to the filtrate, stir III, let stand I, pour out the supernatant to obtain the oily substance in the lower layer; S4, adding ammonium sulfate solution to the oily substance, stirring IIII, standing II, pouring out the supernatant to obtain the oily substance at the bottom, drying to obtain the protamine sulfate.

2. The preparation method according to claim 1, characterized in that: In step S1, the salt water is a sodium chloride aqueous solution; Preferably, the concentration of the brine is 1 mol / L to 3 mol / L; The amount of salt water added is 1.5 to 5 times the weight of the mature testicular tissue of the aquatic organism; Preferably, in step S1, stirring I is performed at 20 to 30° C. for 0.5 to 3.5 hours.

3. The preparation method according to claim 1, characterized in that: In step S2 and step S3, the molecular weight of the polyethyleneimine is independently selected from 1000 to 25000.

4. The preparation method according to claim 1, characterized in that: In step S2, the mass concentration of polyethyleneimine in the final solution is 0.2-1%.

5. The preparation method according to claim 1, characterized in that: In step S2, the amount of water added is to adjust the final concentration of the brine to 1-2 mol / L; Preferably, the amount of water added is such as to adjust the final concentration of the brine to 1.5-1.8 mol / L.

6. The preparation method according to claim 1, characterized in that: In step S2, stirring II is performed at 50 to 80°C for 0.5 to 1.5 hours.

7. The preparation method according to claim 1, characterized in that: In step S3, the mass concentration of polyethyleneimine in the final solution is 0.2-0.96%.

8. The preparation method according to claim 1, characterized in that: In step S3, the amount of water added is to adjust the final concentration of the brine to 0.5-1 mol / L.

9. The preparation method according to claim 1, characterized in that: In step S3, stirring III is performed at 20 to 30°C for 0.5 to 1.5 hours.

10. The preparation method according to claim 1, characterized in that: In step S4, the mass concentration of the ammonium sulfate solution is 20-30%; The amount of ammonium sulfate solution added is 1.5 to 2.5 times the weight of the oil; Preferably, in step S4, stirring IIII is performed at 50-80° C. for 0.5-2 hours.

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