Green preparation method of high-purity trigonelline

Abstract

The invention relates to the technical field of natural product extraction, and discloses a green preparation method of high-purity trigonelline. The method comprises the following steps: (1) performing first treatment on a degreased fenugreek seed raw material in an ethanol water solution under 400-500MPa, and removing a solvent to obtain a mixture I; (2) carrying out second treatment on the mixture I in an aqueous two-phase system, and separating to obtain an upper phase solution rich in trigonelline; and (3) sequentially performing ultrafiltration membrane impurity removal, nanofiltration membrane concentration, resin purification, concentration crystallization and drying on the upper-phase solution to obtain the high-purity trigonelline. The method provided by the invention is efficient and low in consumption, the obtained trigonelline is high in purity, and high-selectivity extraction and purification of the trigonelline are realized.

Description

A green preparation method for high-purity trigonelline Technical Field

[0001] This invention relates to the field of natural product extraction technology, specifically to a green preparation method for high-purity trigonelline. Background Technology

[0002] Trigonelline (chemical name N-methylnicotinic acid inner salt) is a quaternary ammonium alkaloid isolated from the seeds of the legume *Trigonella foenum-graecum* L., possessing both hydrophilic and weakly basic molecular structures. Modern pharmacological studies have shown that this compound can significantly enhance insulin sensitivity by activating the AMPK signaling pathway, reducing fasting blood glucose levels in type 2 diabetic mice by more than 35%; simultaneously, by inhibiting the NF-κB inflammatory pathway, it reduces LPS-induced TNF-α secretion from macrophages by up to 62.3%. In terms of neuroprotection, trigonelline can penetrate the blood-brain barrier, reducing neuronal apoptosis induced by β-amyloid protein 25-35 fragments, and improving cognitive ability scores in Alzheimer's disease model rats by 40%. Due to its multiple biological activities and high safety profile from natural sources, trigonelline has been included in the 11.0 edition of the European Pharmacopoeia's list of active plant ingredients, with global market demand growing at an average annual rate of 12.5%.

[0003] Currently, industrial extraction mainly relies on organic solvent reflux and ultra-high pressure assisted extraction (UHPE). The former typically uses a 60-80 vol% ethanol aqueous solution, refluxed at 70-80℃ for 2-3 extractions (solid-liquid ratio 1:10-15), but has significant drawbacks: firstly, trigonelline has poor selectivity in ethanol-water solutions, resulting in impurities such as sugars and proteins accounting for over 55% of the crude extract, with typical purity only 38-42% as determined by HPLC; secondly, each ton of raw material consumes over 1.2 tons of ethanol, and solvent recovery energy accounts for 45% of production costs. While UHPE (300-600 MPa) can improve cell disruption efficiency and shorten extraction time to 30 minutes, the equipment investment cost is up to three times that of traditional methods, and high pressure treatment easily leads to the degradation of heat-sensitive alkaloids, with the trigonelline isomer content in the product reaching up to 7.8%. More importantly, the above methods all require multiple purification steps such as silica gel column chromatography or preparative HPLC to meet pharmaceutical standards (purity ≥90%), and the overall process takes more than 8 hours with a yield of less than 10%.

[0004] CN103755630A discloses a method for enriching and purifying trigonelline in fenugreek using ultra-high pressure technology and macroporous resin adsorption and column chromatography coupling technology, but there are problems such as many impurities in the crude product and difficulties in subsequent purification.

[0005] Aqueous two-phase extraction (ATPE), a technique that has emerged in recent years, is considered an alternative due to its use of water as a continuous phase, mild conditions, and environmental friendliness. A typical system is the polyethylene glycol (PEG) / ammonium sulfate system, which achieves separation by adjusting the polymer molecular weight (PEG 1000-6000) and the inorganic salt concentration (10-25%), utilizing the difference in the partition coefficient (K) of the solute between the two phases.

[0006] However, when applied to the extraction of trigonelline, the aqueous two-phase extraction technology faces three major bottlenecks: First, the quaternary ammonium structure of trigonelline makes it easy to form micelles at the interface between the two phases, resulting in a phase separation time of more than 45 minutes (compared to only 15 minutes in the traditional system), and the phase volume ratio fluctuates between 0.8 and 1.5; Second, the critical concentration for phase formation in the PEG / ammonium sulfate system is sensitive to temperature, and the partition coefficient K value of trigonelline drops sharply from 3.7 to 1.2 in the range of 25℃ to 40℃, with the recovery rate of the target component deviating by more than 20%; Third, trigonelline saponin B coexisting in the seeds competes with the alkaloids for binding to the hydrophilic segments of PEG, causing the K value to drop by 38%, requiring the addition of salting-out agents to compensate, which in turn triggers emulsification.

[0007] Therefore, it is of great significance to develop a highly stable, low-cost aqueous two-phase extraction process suitable for the extraction of trigonelline. Summary of the Invention

[0008] The purpose of this invention is to overcome the problems in the prior art where trigonelline extraction using ultra-high pressure requires multiple purification steps such as silica gel column chromatography or preparative HPLC to meet pharmaceutical standards, and the problems of prolonged phase separation time, large deviation in target component recovery rate, low K value, and easy emulsification when using polyethylene glycol / ammonium sulfate aqueous two-phase extraction technology to extract trigonelline.

[0009] To achieve the above objectives, the present invention provides a method for preparing high-purity fenugreekine, the method comprising the following steps: (1) under 400-500 MPa, defatted fenugreek seed raw material is subjected to a first treatment in an aqueous ethanol solution with a concentration of 40-60 vol% to remove the solvent and obtain mixture I; wherein the crude fat content of the defatted fenugreek seed raw material does not exceed 1 wt%; (2) under 35-45°C, the mixture I is subjected to a second treatment in an aqueous two-phase system to separate an upper phase solution rich in fenugreekine; wherein the aqueous two-phase system is composed of phosphate buffer, polyethylene glycol and ammonium sulfate; (3) the upper phase solution is subjected to ultrafiltration membrane impurity removal, nanofiltration membrane concentration, resin purification, concentration crystallization and drying in sequence to obtain high-purity fenugreekine.

[0010] Compared with the prior art, the present invention has at least the following advantages: (1) The preparation method provided by the present invention creates favorable conditions for efficient aqueous two-phase extraction through ultra-high pressure, realizing that no further purification is required after ultra-high pressure treatment, and finally achieving the purpose of selective extraction and in-situ removal of impurities: First, ultra-high pressure treatment can efficiently and selectively destroy the cell wall structure of fenugreek seeds through static pressure energy, making it easier to release the target component fenugreekine. At the same time, this physical force can promote the dissociation of fenugreekine from the complexes of some polysaccharides, proteins and other impurities. Degreasing with ethyl acetate can remove a large amount of fat-soluble impurities in situ, reducing the burden of subsequent purification from the source.

[0011] Secondly, cell wall disruption significantly enhances the penetration efficiency of subsequent extractants. Studies have shown that ultra-high pressure treatment can increase the penetration efficiency of subsequent extractants by 3.2 times. This means that the target components can enter the aqueous two-phase system more quickly and thoroughly, while also reducing impurities introduced by excessively extending the extraction time for full extraction, thereby improving the purity of the initial extract.

[0012] Finally, after ultra-high pressure and aqueous two-phase extraction, the macromolecular impurities in the extract were significantly reduced. At this point, only a 10 kDa ultrafiltration membrane was needed to remove residual macromolecular proteins and polysaccharide polymers, and a 200 Da nanofiltration membrane was needed for desalting and concentration to achieve good purification results. Subsequent selective adsorption with macroporous adsorption resin (AB-8) further purified the product to a purity >90%.

[0013] (2) The aqueous two-phase system provided by the present invention provides an ideal separation environment for the components after ultra-high pressure treatment: ultra-high pressure treatment releases a large amount of intracellular contents, at which time a mild and highly selective separation environment is crucial. The specific aqueous two-phase system (PEG / ammonium sulfate-phosphate buffer) of the present invention, with its aqueous and mild properties, can well accommodate these released components. By precisely controlling the phase-forming components, the temperature, pH and ionic strength of the system, and optimizing the molecular weight, concentration and centrifugation conditions of PEG, trigonelline can be selectively enriched in the upper phase, while many water-soluble impurities (such as sugars and some proteins) are distributed in the lower phase or removed by interfacial adsorption, thus achieving preliminary purification and concentration, solving the problems of prolonged phase separation time and emulsification, stabilizing the partition coefficient (K) and improving the recovery rate. Detailed Implementation

[0014] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0015] As mentioned above, the present invention provides a method for preparing high-purity fenugreekine, which includes the following steps: (1) under 400-500 MPa, defatted fenugreek seed raw material is subjected to a first treatment in an aqueous ethanol solution with a concentration of 40-60 vol% to remove the solvent and obtain mixture I; wherein the crude fat content of the defatted fenugreek seed raw material does not exceed 1 wt%; (2) under 35-45℃, the mixture I is subjected to a second treatment in an aqueous two-phase system to separate an upper phase solution rich in fenugreekine; wherein the aqueous two-phase system is composed of phosphate buffer, polyethylene glycol and ammonium sulfate; (3) the upper phase solution is subjected to ultrafiltration membrane impurity removal, nanofiltration membrane concentration, resin purification, concentration crystallization and drying in sequence to obtain high-purity fenugreekine.

[0016] Preferably, in step (1), the solvent removal is performed by filtration.

[0017] Preferably, in step (1), the defatted fenugreek seed raw material is a product obtained by soaking and defatting fenugreek seed powder with ethyl acetate, wherein the particle size distribution D90 of the fenugreek seed powder is ≤180μm, and the lipid removal rate achieved by the soaking and defatting is ≥92wt%. Studies have found that under this preferred condition, it is beneficial to achieve a cell wall breakage rate of ≥85% while avoiding fine powder agglomeration.

[0018] Preferably, the soaking and defatting process includes: adding fenugreek seed powder and ethyl acetate with a water content of ≤0.1wt% into a pressure-resistant extraction tank at a material-to-liquid ratio of 1:12-16 (g / mL), and soaking for 60 minutes at a stirring speed of 200 rpm and a temperature of 25°C to fully dissolve fat-soluble impurities such as fatty acids and sterols.

[0019] In a preferred embodiment, in step (1), the pressure increase rate is 100 MPa / s, and the first processing time is 3-5 min.

[0020] Preferably, in step (1), the first process is performed in an ultra-high voltage device.

[0021] In a preferred embodiment, in step (2), the average molecular weight of the polyethylene glycol is 400-2000, and the molecular weight distribution PDI is ≤1.1.

[0022] More preferably, in step (2), the average molecular weight of the polyethylene glycol is 1500-2000.

[0023] In a preferred embodiment, in step (2), the concentration of polyethylene glycol in the aqueous two-phase system is 15-20 wt%, and the concentration of ammonium sulfate is 10-15 wt%.

[0024] More preferably, in step (2), the aqueous two-phase system is prepared by a method including the following steps: polyethylene glycol, ammonium sulfate and phosphate buffer are mixed at 800 rpm for 30 min, and after standing and phase separation, the phase diagram is measured to ensure that the phase formation region is located in the upper right quadrant of the binode line, thus obtaining the aqueous two-phase system.

[0025] Preferably, in step (2), the phosphate buffer solution is prepared by dissolving Na2HPO4 and KH2PO4 in water.

[0026] In a preferred embodiment, in step (2), the total phosphate concentration of the phosphate buffer is 0.1 mol / L, the pH is 6.0-7.0, and the content of the phosphate buffer in the aqueous two-phase system is 65-75 wt%.

[0027] Preferably, the ionic strength I of the phosphate buffer solution is 0.25 ± 0.02.

[0028] In a preferred embodiment, in step (2), the weight ratio of the mixture I to the aqueous two-phase system is 1:3-5.

[0029] In a preferred embodiment, in step (2), the second process is performed under oscillating conditions and at least satisfies the following conditions: amplitude of 20-40 mm, frequency of 90-150 times / min, and time of 20-40 min.

[0030] In a preferred embodiment, in step (2), the separation is carried out by centrifugation, with a g value of 3000×g and a time of 5-20min.

[0031] Preferably, the centrifugal separation is performed using a horizontal screw centrifuge.

[0032] In a preferred embodiment, in step (3), the ultrafiltration membrane impurity removal is performed using a ceramic membrane module with a pore size of 0.01 μm and a corresponding molecular weight cutoff of 10 kDa.

[0033] In a preferred embodiment, in step (3), the molecular weight cutoff of the nanofiltration membrane concentration is 200 Da.

[0034] Preferably, the ultrafiltration membrane impurity removal further includes: cross-flow filtration at a transmembrane pressure difference of 0.3-0.4 MPa, a flow rate of 15 L / min, and a membrane flux maintained at not less than 80 LMH, to remove PEG-protein complexes and residual polysaccharides, thereby obtaining the ultrafiltration permeate.

[0035] Preferably, in step (3), a polyamide spiral wound nanofiltration membrane is used to concentrate the nanofiltration membrane to 1 / 5 of the original volume of the ultrafiltration permeate, and the operating pressure is 1.8-2.2 MPa.

[0036] Preferably, in step (3), the resin purification includes: passing the resin through an AB-8 type macroporous resin column at a flow rate of 2 BV / h, rinsing with 3 BV of pure water after adsorption saturation, and then eluting with 20 vol% ethanol aqueous solution at 1.5 BV / h, and collecting the elution peak with a UV detection wavelength of 265 nm.

[0037] More preferably, the AB-8 type macroporous resin column has a column height-to-diameter ratio of 5:1 and a column packing volume of 10 BV.

[0038] Preferably, in step (3), the concentration and crystallization includes: concentrating the eluent under reduced pressure at a water bath temperature of 40°C and a vacuum degree of -0.095MPa to obtain a supersaturated concentrate, and then precipitating trigonelline crystals by gradient crystallization.

[0039] More preferably, in the concentrated crystallization, the gradient crystallization method includes: adding 75-90 vol% ethanol aqueous solution to the concentrate, dissolving it at 50°C, cooling it to 4°C at a rate of 0.5°C / min, and allowing it to stand for 24 hours to crystallize, thereby obtaining trigonelline crystals, wherein the amount of seed crystals added is 0.1 wt%.

[0040] Preferably, in step (3), the drying includes: filtering the trigonelline crystals through a Buchner funnel, washing them twice with an 85 vol% pre-cooled ethanol aqueous solution, and drying them under vacuum to constant weight to obtain white needle-like crystals, which are high-purity trigonelline.

[0041] More preferably, in the drying process, the filter membrane of the Buchner funnel used for filtration has a pore size of 0.45 μm.

[0042] More preferably, in the drying process, the vacuum drying conditions at least satisfy: a temperature of 40°C and a vacuum degree of -0.09 MPa.

[0043] The present invention will be described in detail below through examples. Unless otherwise specified, the raw materials used are all commercially available products.

[0044] Ethyl acetate: chromatographic grade, water content ≤0.1wt%; Ultra-high pressure equipment: UHP-600, pressure range 0-700MPa; Polyethylene glycol: average molecular weight 2000, molecular weight distribution PDI ≤1.1; Ammonium sulfate: pesticide residue grade, purity ≥99.5%; Horizontal decanter centrifuge: LW450×2000; HPLC column specifications: C18, 5μm, 4.6×250mm; Spiral wound nanofiltration membrane: material: polyamide, molecular weight cutoff 200Da; Ceramic membrane module: pore size 0.01μm, corresponding molecular weight cutoff 10kDa; AB-8 macroporous resin column: column height-to-diameter ratio 5:1, packing volume 10BV.

[0045] Phosphate buffer: The solution was prepared by dissolving Na2HPO4 and KH2PO4 in water. The solutions were: Phosphate Buffer I: total phosphate concentration 0.1 mol / L, pH 6.5, ionic strength I 0.25 ± 0.02; Phosphate Buffer II: total phosphate concentration 0.1 mol / L, pH 5.5, ionic strength I 0.25 ± 0.02. For the preparation of defatted fenugreek seed raw material: fenugreek seeds were pulverized to a particle size distribution D90 of 180 μm using a stainless steel universal pulverizer (sieve aperture 0.18-0.15 mm). The resulting fenugreek seed powder was then mixed with ethyl acetate at a ratio of 1:12-16 (g / mL) in a pressure-resistant extraction tank and soaked at 200 rpm and 25℃ for 60 min to fully dissolve fat-soluble impurities such as fatty acids and sterols, resulting in defatted fenugreek seed raw material. The final defatting process achieved a lipid removal rate ≥92 wt%.

[0046] Aqueous two-phase system: Preparation of aqueous two-phase system I: Polyethylene glycol, ammonium sulfate and phosphate buffer (phosphate buffer I) were mixed at 800 rpm for 30 min. After standing and phase separation, the phase diagram was measured to ensure that the phase formation region was located in the upper right quadrant of the binode line (critical phase formation concentration: PEG 14.8 wt% / ammonium sulfate 9.3 wt%), resulting in aqueous two-phase system I with polyethylene glycol concentration of 18 wt%, ammonium sulfate concentration of 12 wt%, and phosphate buffer content of 70 wt%.

[0047] Preparation of Aqueous Two-Phase System II: The preparation method of Aqueous Two-Phase System I was followed, except that the amounts of substances were adjusted so that the final aqueous two-phase system contained polyethylene glycol at a concentration of 20 wt%, ammonium sulfate at a concentration of 10 wt%, and phosphate buffer at a concentration of 70 wt%. Preparation of Aqueous Two-Phase System III: The preparation method of Aqueous Two-Phase System I was followed, except that phosphate buffer II was used.

[0048] Example 1 This example illustrates the method for preparing high-purity fenugreek alkaloids provided by the present invention, which is carried out according to the following steps: (1) transferring defatted fenugreek seed raw material to an ethanol aqueous solution with a concentration of 50 vol%, and then in an ultra-high pressure device, increasing the pressure to 450 MPa at a pressurization rate of 100 MPa / s for a first treatment of 4 min, and filtering to remove the solvent to obtain mixture I; wherein, the crude fat content of the defatted fenugreek seed raw material does not exceed 1 wt%; (2) under the condition of oscillation at 40±1℃ using an eccentric wheel oscillator with an amplitude of 30 mm and a frequency of 120 times / min, using a dosage of 1:4 The mixture I was subjected to a second treatment in a two-phase system (two-phase system I) for 30 min, and then centrifuged using a horizontal screw centrifuge (g value 3000×g, time 10 min) to obtain an upper phase solution rich in trigonelline. At this time, the trigonelline partition coefficient K≥5.2 and the phase volume ratio was 1.2-1.4; (3) The upper phase solution was subjected to the following operations in sequence: ultrafiltration membrane impurity removal: using a ceramic membrane module, cross-flow filtration was performed at a transmembrane pressure difference of 0.3-0.4 MPa, a flow rate of 15 L / min, and the membrane flux was maintained at not less than 80 LMH to remove PEG-protein complex and residual polysaccharides to obtain ultrafiltration permeate; nanofiltration membrane concentration The ultrafiltration permeate was concentrated to 1 / 5 of its original volume using a polyamide spiral-wound nanofiltration membrane with a molecular weight cutoff of 200 Da at an operating pressure of 1.8-2.2 MPa. Resin purification: The solution was passed through an AB-8 macroporous resin column at a flow rate of 2 BV / h. After adsorption saturation, it was first rinsed with 3 BV of pure water, then eluted with a 20 vol% ethanol aqueous solution at a flow rate of 1.5 BV / h. The elution peak at a UV detection wavelength of 265 nm was collected to obtain the eluent. Concentration and crystallization: The eluent was concentrated under reduced pressure at a water bath temperature of 40℃ and a vacuum degree of -0.095 MPa to obtain a supersaturated concentrate (trigonelline content ≥35 wt%). Then, 85% ethanol was added to the concentrate. A vol% ethanol aqueous solution was dissolved at 50℃ and then cooled to 4℃ at a rate of 0.5℃ / min. After standing for 24 hours, crystallization was obtained to obtain trigonelline crystals, with a seed crystal addition of 0.1wt%. Drying: The trigonelline crystals were filtered through a Buchner funnel (filter membrane pore size 0.45μm), washed twice with 85 vol% pre-cooled ethanol aqueous solution, and vacuum dried (temperature 40℃, vacuum degree -0.09MPa) to constant weight to obtain white needle-like crystals (bulk density 0.42g / cm³, angle of repose ≤30°), which is high-purity trigonelline, named P1, with a yield of 9.8wt% and a purity of 92.7wt% as determined by HPLC.

[0049] Example 2 This example uses a similar method to Example 1, except that in step (2), the aqueous two-phase system used is aqueous two-phase system II. Finally, high-purity trigonelline was obtained, named P2, with a yield of 10.1 wt% and a purity of 91.5 wt% as determined by HPLC.

[0050] Example 3 This example uses a similar method to Example 1, except that in step (21), the aqueous two-phase system used is aqueous two-phase system III. Finally, high-purity trigonelline was obtained, named P3, with a yield of 8.9 wt% and a purity of 86.9 wt% as determined by HPLC.

[0051] Comparative Example 1 This comparative example was carried out using a method similar to that of Example 1, except that in step (1), the pressure of the first treatment was 0.1 MPa; finally, trigonelline was obtained, named DP1, with a yield of 7.6 wt% and a purity of 83.5 wt% as determined by HPLC.

[0052] Comparative Example 2: This comparative example used a conventional method to extract trigonelline from fenugreek seeds, specifically including: S1, pulverizing fenugreek seeds to an average volume diameter of 0.15-0.17 mm, adding 8 times the volume of 80 vol% ethanol aqueous solution, refluxing at 85°C twice, 1 h each time, filtering the obtained extracts, combining them, concentrating, and vacuum drying at 60°C to obtain crude trigonelline extract; S2, dissolving the crude trigonelline extract obtained in step S1 in petroleum ether, adding an appropriate amount of silica gel and stirring, drying, pulverizing to an average volume diameter of 0.20-0.25 mm, dry loading, and chromatography on a 200-mesh industrial-grade silica gel column; S3, using acetone and petroleum ether at a volume ratio of 1:1... Gradient elution was performed under the conditions of 1:2:1, 3:1, 4:1, 5:1, 6:1, and 7:1. Thin-layer chromatography was used for follow-up detection. The eluents of trigonelline at each stage were collected, concentrated under reduced pressure, and the solvent was recovered. S4. The obtained concentrate was dried under vacuum at 60°C. S5. The product obtained in step S4 was dissolved in 10 times its volume of 80 vol% ethanol aqueous solution, and then concentrated to one-tenth of the original volume. It was left overnight to obtain crystals. After separating the crystals, the remaining mother liquor was dissolved in 60 vol% ethanol aqueous solution, concentrated again, and recrystallized. Finally, the crystals obtained from the two processes were combined to obtain trigonelline, named DP2, with a yield of 3.8 wt% and a purity of 64.7 wt% as determined by HPLC.

[0053] The results above demonstrate that the method provided by this invention is not only highly efficient and low-consumption, but also yields high-purity trigonelline, achieving highly selective extraction and purification of trigonelline. The ultra-high pressure-aqueous two-phase synergistic extraction improves the partition coefficient of trigonelline in the aqueous two-phase system (K≥5.2), and the product purity is increased by more than 40% compared to traditional methods. Using a polyethylene glycol / ammonium sulfate system instead of organic solvents reduces toxicity and improves the phase separation rate.

[0054] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for preparing high-purity trigonelline, characterized in that, The method includes the following steps: (1) under 400-500 MPa, defatted fenugreek seed raw material is subjected to a first treatment in an ethanol aqueous solution with a concentration of 40-60 vol% to remove the solvent and obtain mixture I; wherein the crude fat content of the defatted fenugreek seed raw material does not exceed 1 wt%; (2) under 35-45℃, the mixture I is subjected to a second treatment in an aqueous two-phase system to separate an upper phase solution rich in fenugreekine; wherein the aqueous two-phase system is composed of phosphate buffer, polyethylene glycol and ammonium sulfate; (3) the upper phase solution is subjected to ultrafiltration membrane impurity removal, nanofiltration membrane concentration, resin purification, concentration crystallization and drying in sequence to obtain high-purity fenugreekine.

2. The method according to claim 1, characterized in that, In step (1), the pressure increase rate is 100 MPa / s, and the first processing time is 3-5 min.

3. The method according to claim 1 or 2, characterized in that, In step (2), the average molecular weight of the polyethylene glycol is 400-2000, and the molecular weight distribution PDI is ≤1.

1.

4. The method according to claim 1 or 2, characterized in that, In step (2), in the aqueous two-phase system, the concentration of polyethylene glycol is 15-20 wt%, and the concentration of ammonium sulfate is 10-15 wt%.

5. The method according to claim 1 or 2, characterized in that, In step (2), the total phosphate concentration of the phosphate buffer is 0.1 mol / L, the pH is 6.0-7.0, and the content of the phosphate buffer in the aqueous two-phase system is 65-75 wt%.

6. The method according to claim 1 or 2, characterized in that, In step (2), the weight ratio of the mixture I to the aqueous two-phase system is 1:3-5.

7. The method according to claim 1 or 2, characterized in that, In step (2), the second process is carried out under oscillating conditions and at least meets the following requirements: amplitude of 20-40 mm, frequency of 90-150 times / min, and time of 20-40 min.

8. The method according to claim 1 or 2, characterized in that, In step (2), the separation is carried out by centrifugation, with a g value of 3000×g and a time of 5-20min.

9. The method according to claim 1 or 2, characterized in that, In step (3), the ultrafiltration membrane impurity removal is performed using a ceramic membrane module with a pore size of 0.01 μm and a corresponding molecular weight cutoff of 10 kDa.

10. The method according to claim 1 or 2, characterized in that, In step (3), the molecular weight cutoff of the nanofiltration membrane concentration is 200 Da.