Method for efficiently separating stigmasterol monomer by using amphiphilic glycoside derivative

Abstract

The invention relates to the technical field of stigmasterol monomer separation, in particular to a method for efficiently separating a stigmasterol monomer by using an amphiphilic glucoside derivative, which comprises the following steps: S1, capturing stigmasterol, S2, separating out a stigmasterol-glucoside compound, S3, obtaining a stigmasterol crude product, and S4, refining stigmasterol. The amphiphilic glycoside derivative is utilized to realize double recognition of a beta-CD cavity and a thioester bond, the stigmasterol binding amount is obviously higher than that of beta-sitosterol, the separation factor reaches 3.8 or above, and the separation effect is better.

Description

Technical Field

[0001] This invention relates to the field of stigmasterol monomer separation technology, specifically to a method for efficiently separating stigmasterol monomers using amphiphilic glycoside derivatives. Background Technology

[0002] Currently, the separation methods for stigmasterol monomers mainly rely on differences in the solubility of each sterol monomer in organic solvents, differences in boiling points under high vacuum conditions, and differences in adsorption forces. However, because stigmasterol shares a similar cyclopentane-polyhydrophenanthrene core structure with β-sitosterol and campesterol, with only slight differences in the position and length of the side chain double bonds, this high similarity means that traditional solvent crystallization methods require more than nine recrystallizations to achieve 95% purity. Furthermore, the process is cumbersome, uses large amounts of solvent, and has low product yields. While chromatographic methods (such as Prep-HPLC) offer high purity, their scale is limited, suitable only for laboratory settings, and not feasible for industrialization. Emerging supercritical CO2 extraction technology involves high equipment investment and offers limited improvement in product purity, restricting its industrial application. Summary of the Invention

[0003] To address the above problems, this invention provides a method for efficiently separating stigmasterol monomers using amphiphilic glycoside derivatives, comprising the following steps: S1, Capture Stigmasterol At 23~27℃, the amphiphilic glycoside derivative β-CD-PNIPAM-MPA was dissolved in deionized water at a ratio of 1g:60~75mL. Then, phytosterol was added at a mass ratio of 1:8~12 between the amphiphilic glycoside derivative and phytosterol. The pH was adjusted to 6.5~7.0, and the mixture was stirred for 2~2.5h to capture stigmasterol. S2, precipitation of stigmasterol-glycoside complex The stigmasterol obtained from S1 was heated to 40-50℃ at a rate of 1℃ / min and kept at that temperature for 15-30 min. It was then centrifuged at 5500-6000 rpm for 15-20 min to separate the solid and liquid phases and precipitate the stigmasterol-glycoside complex. S3. Obtain crude stigmasterol product The stigmasterol-glycoside complex was washed 2-3 times with an ethanol aqueous solution of 13-15% by mass at 40-50℃. The mixture was centrifuged to obtain a centrifugal liquid and a solid phase. The centrifugal liquid was cooled, kept warm, filtered, and dried to obtain crude stigmasterol. The solid phase was dissolved in deionized water at 25℃, filtered to remove impurities, and then freeze-dried at -45 to -55℃ and a vacuum degree ≤10Pa for 4-8 hours to recover β-CD-PNIPAM-MPA for future use. S4, refined stigmasterol The crude stigmasterol was added to 98wt% n-butanol at a ratio of 1g:2.5~4.0mL and recrystallized 2~3 times to obtain the stigmasterol monomer. Explanation: Separation principle: ① Low-temperature capture stage (25℃) Molecular recognition: β-CD cavity encapsulates stigmasterol sterol core, and thioester bond forms covalent bond with 3-OH, a dual effect to enhance selectivity (stigmasterol adsorption capacity > 65 mg / g, β-sitosterol < 20 mg / g). Dispersion state: PNIPAM hydrophilic extension ensures uniform dispersion of the complex in the aqueous phase, avoiding aggregation.

[0004] ②Heating precipitation stage (45℃) phase change driven separation: PNIPAM chain dehydrates and shrinks, causing the entire molecule to aggregate and precipitate, stigmasterol-glycoside complex precipitates (particle size 1–5μm, easy to centrifuge), β-sitosterol is retained in the liquid phase due to weak binding.

[0005] ③ Dynamic bond breaking during stigmasterol release: 15% ethanol (pH 4.0) weakens the thioester bond, releasing stigmasterol (release rate > 95%). Simultaneously, PNIPAM extends in ethanol, promoting the dissociation of the complex. Recrystallization utilizes the difference in solubility of stigmasterol and other sterols in n-butanol to achieve the separation of stigmasterol. The purity of crude stigmasterol reaches 75%, and the purity of the purified stigmasterol monomer can reach 95%. This method can recover and reuse β-CD-PNIPAM-MPA from the preparation process, reducing raw material consumption and energy input, further improving sustainability and environmental friendliness.

[0006] Further, in S1, the preparation method of the amphiphilic glycoside derivative β-CD-PNIPAM-MPA is as follows: S1-1. β-Cyclodextrin was dissolved in anhydrous DMSO at a mass ratio of 1 g: 8-12 mL under conditions of 23-27℃. The solution was cooled to 0℃ by purging with nitrogen. Then, 2-bromoisobutyryl bromide was added dropwise at a molar ratio of 1:6-9 (β-cyclodextrin primary hydroxyl group: 2-bromoisobutyryl bromide). The mixture was stirred for 2-2.5 h, centrifuged at 7500-8500 rpm for 5-10 min to collect the precipitate, washed 2-4 times with cold diethyl ether at 0-5℃, and centrifuged again at 7500-8500 rpm for 5-10 min to collect the precipitate. The precipitate was then vacuum dried at 55-65℃ and -0.06--0.095 MPa for 4-8 h to obtain β-bromodiphenyl ether (β-CD). The degree of substitution of β-CD was 6.0-8.0. S1-2. Mix β-CD bromide, NIPAM, CuBr, and PMDETA according to the molar ratio of Br atoms in β-CD bromide : NIPAM : CuBr : PMDETA of 1:45 - 55:0.1:0.12 to obtain a mixture. Then dissolve the mixture in a methanol-aqueous solution with a mass fraction of 75 - 80% according to the ratio of β-CD bromide to the methanol-aqueous solution of 1 g:5 - 10 mL. Purge with nitrogen for 25 - 30 min, and then react at 30 - 40 °C for 5.5 - 6.5 h. Purify by membrane dialysis to remove unreacted monomers, and treat at -40 - -50 °C and 0.1 - 0.15 mbar for 20 - 24 h to obtain β-CD-PNIPAM; S1-3. Dissolve β-CD-PNIPAM and 3-mercaptopropionic acid in the PBS buffer solution with pH = 7.4 according to the molar ratio of Br atoms in β-CD-PNIPAM : 3-mercaptopropionic acid of 1:4 - 6 and the ratio of β-CD-PNIPAM to the PBS buffer solution of 1 g:5 - 10 mL. Stir at 23 - 27 °C for 22 - 24 h, purify by dialysis and then freeze-dry to obtain an amphiphilic glycoside derivative, denoted as β-CD-PNIPAM-MPA; Note: The poly(N-isopropylacrylamide) (PNIPAM) chain has the property of lower critical solution temperature (LCST, about 32 °C). At low temperature (<32 °C), the PNIPAM chain is hydrophilic and extended, making the whole derivative soluble in water; at high temperature (>32 °C), the PNIPAM chain dehydrates and collapses, changing from hydrophilic to hydrophobic, resulting in hydrophobic aggregation between molecules and precipitation from water; Capture stage: At room temperature (<LCST), mix the derivative with an extract containing stigmasterol, and the β-CD cavity encapsulates stigmasterol; Separation stage: Just heat the system to above LCST (such as 40 °C), and the whole "β-CD-PNIPAM-MPA-stigmasterol" complex will form large aggregated particles, which can be easily separated by standing or low-speed centrifugation. This completely replaces the expensive and time-consuming chromatographic column, greatly improving the separation efficiency, reducing energy consumption and costs; After separation, wash the precipitate with a small amount of clear water or solvent at low temperature, the PNIPAM chain re-stretches, the complex dissolves and releases pure stigmasterol monomers, and at the same time the material itself is regenerated and can be recycled. And the 3-mercaptopropionic acid (MPA) introduced by the thiol-bromine click chemical reaction provides a carboxyl group at the end of the molecule. The presence of the carboxyl group allows fine-tuning of the hydrophilicity and hydrophobicity of the whole molecule by adjusting the pH value, further optimizing its thermosensitive behavior and interaction with stigmasterol.

[0007] Further, in S1, the pH adjustment method is: Drop in a phosphate buffer solution with a concentration of 0.1 - 0.2 M at a dropping rate of 5 - 10 mL / min; Note: Slowly adding the buffer solution avoids excessively high local concentrations, preventing the target stigmasterol or β-CD-PNIPAM-MPA from being degraded or inactivated due to extreme local pH during the adjustment process.

[0008] Furthermore, in S3, the parameters for cooling, heat preservation, filtration, and drying are as follows: natural cooling to 2~4℃, then heat preservation for 1.5~2h, centrifugal filtration at a speed of 5500~6000rpm, and drying at a temperature of 85~95℃ for 4~8h. Note: The above method separates stigmasterol that is dispersed and dissolved in ethanol and water.

[0009] Further, in S4, the recrystallization method is as follows: stirring and heating at a rate of 5~10℃ / min to 60~68℃ and holding for 15~20min, then naturally cooling to 45~50℃ and holding for 3.5~4h; repeating the stirring and heating and natural cooling steps 1~2 times; Explanation: Recrystallization utilizes the principle of hot dissolution and cold precipitation, taking advantage of the different solubilities of stigmasterol and sitosterol at different temperatures to separate them. Slow cooling and long-term holding promote the orderly growth of crystals and reduce the possibility of impurities (sitosterol molecules) being trapped in the crystal lattice (reducing lattice defects). Holding at 45~50℃ for 3.5~4 hours provides sufficient and stable time for crystal growth, allowing stigmasterol crystals to grow slowly and orderly, forming large crystals with complete crystal forms and higher purity. Large crystals are easier to filter and contain fewer impurities (mother liquor and possibly co-crystallized sitosterol).

[0010] Furthermore, in S1-3, the freeze-drying process is as follows: first, the material temperature is lowered to -45℃ by vacuuming with a freeze dryer, and then increased by 5-10℃ every 1-3 hours. When the temperature reaches above 0℃, it is increased by 5-10℃ every 2-4 hours. When the temperature shown by the upper, middle and lower temperature probes reaches 40℃ and is maintained for more than 12 hours, the material can be discharged. Note: 0~-45℃ is the primary drying temperature: when the material temperature drops to -45℃, the free water has frozen into ice. In a high vacuum environment, a slow, gradient heating process causes the free water ice to sublimate, thus removing the free water.

[0011] Secondary drying occurs above 0℃: Under vacuum conditions, after all free water is removed, primary drying ends. The secondary drying stage then begins, removing adsorbed bound water from the material. This is primarily achieved through heating to desorb the bound water, using a gradient temperature increase to desorb bound water at different energy levels.

[0012] Furthermore, in S1-2, the membrane dialysis purification uses a membrane with a molecular weight cutoff of 3500 Da. The purification method is as follows: the liquid to be purified is poured into the feed tank, the feed pressure is controlled to be ≤0.3MPa, and the discharge pressure is ≤0.2MPa. When the retentate in the ceramic membrane tube cannot circulate, purified water is added. The amount of purified water added is 3 times the amount of the retentate added in 3 to 4 separate additions. Explanation: Unlike traditional methods where the feed solution is vertically directed towards the membrane surface, the feed pressure > discharge pressure allows the feed solution to flow parallel to the membrane surface at high speed. This continuously washes the membrane surface, preventing trapped large molecules from forming a dense gel layer (concentration polarization) that clogs the membrane pores. This maintains a high filtration rate and extends the membrane's lifespan. Each time, adding one volume of water and concentrating the solution removes approximately 50% of the remaining small molecule impurities. Repeating this process 3-4 times, for a total of 3 times the volume, can reduce the residual amount of small molecule impurities to a few percent or even lower than the initial concentration (for example, after 3 times the volume of water, the impurity residue can be reduced to approximately (1 / 2)³ = below 12.5%). This is much better than simply concentrating to a very small volume and then resolving it, resulting in higher purity and reducing water consumption and the number of filtration cycles.

[0013] Further, prior to S1-2, the brominated β-CD is loaded onto a carboxylated support; the loading method is as follows: First, magnetic Fe3O4 nanoparticles were synthesized by coprecipitation and then aminated with (3-aminopropyl)triethoxysilane to obtain aminated magnetic nanoparticles. Next, the aminated magnetic nanoparticles were dissolved in anhydrous DMSO at a ratio of 1-3g:200mL, and ultrasonically dispersed for 25-30min to form a suspension. The suspension was then placed in an ice bath at 0-5℃. A 0.2-0.5mol / L succinic anhydride solution was added at a molar ratio of 1:2-3 between the amino groups on the surface of the aminated magnetic nanoparticles and the succinic anhydride solution. The mixture was stirred for 0.5-1h, and then stirred and reacted at 23-25℃ for 12-24h. After standing, the mixture was treated with a 0.3-0.5T NdFeB permanent magnet near the container wall for 5-15min. The supernatant was discarded, and the solid product was collected and washed 2-3 times with anhydrous DMSO to obtain the carboxylated support. The succinic anhydride solution was obtained by dissolving succinic anhydride in anhydrous DMSO. Then, the carboxylated support, β-CD bromide, and catalyst were dispersed in anhydrous DMSO at a mass ratio of 1:1.2~1.7:0.4~0.6:0.04~0.06. The mixture was ultrasonically stirred to form a dispersion, and stirred at 23~25℃ in the dark for 24~48h under nitrogen protection. Finally, the mixture was treated with a neodymium iron boron permanent magnet with a magnetic field strength of 0.3~0.5 T near the container wall for 10~15 min. The supernatant was discarded, the solid product was collected, and washed with DMSO, ethanol, and deionized water in sequence. After vacuum drying at 38~42℃ for 10~12h, β-CD bromide loaded on the carboxylated support was obtained. Explanation: The carboxyl group provides an active site for the subsequent reaction with the hydroxyl group of β-CD. By immobilizing Br-β-CD onto the carboxylated support, the initiation site (-Br) of each Br-β-CD molecule participating in the subsequent ATRP reaction is uniformly exposed and extended from the primary hydroxyl group. As a result, the PNIPAM chains grown in this way have a consistent spatial orientation, avoiding steric hindrance caused by random grafting. The structurally uniform β-CD-PNIPAM-MPA can significantly improve the adsorption capacity and selectivity in the low-temperature capture stage.

[0014] Furthermore, the catalyst is 4-dimethylaminopyridine; Note: The above-mentioned components were selected as catalysts. A small amount of addition can greatly improve the reaction rate. By generating stable acylpyridinium salts, the side reaction of O-acyl isourea rearrangement is effectively avoided, improving the selectivity and specificity of the reaction. This ensures that the activated carboxyl group is mainly used for the formation of the target ester bond, thereby obtaining a higher grafting amount and a purer product.

[0015] Compared with existing technologies, the beneficial effects of this invention are: (1) Existing recrystallization methods are inefficient, require nine recrystallizations, and consume large amounts of solvent. This invention utilizes amphiphilic glycoside derivatives to achieve dual recognition of β-CD cavity and thioester bond. The binding amount of stigmasterol is significantly higher than that of β-sitosterol, and the separation factor reaches more than 3.8. In view of the limitations of laboratory scale in chromatography and the high equipment investment of supercritical methods, this invention only requires conventional stainless steel reactors and solid-liquid separation equipment, which is conducive to industrial scale-up. Moreover, the amphiphilic glycoside derivatives can be recycled and reused, and the overall cost is relatively economical.

[0016] (2) This invention addresses the problem that relying solely on centrifugation in the S2 precipitation step may result in some small aggregates not being completely centrifuged, the centrifugation operation being cumbersome, and the loss of samples during transfer, leading to reduced efficiency. By loading β-CD bromide onto a carboxylated support, this invention achieves instantaneous solid-liquid separation. This separation method is more thorough, faster, and easier to operate, effectively reducing material loss and thus directly increasing the yield of stigmasterol. Furthermore, addressing the problem that during ATRP polymerization and MPA grafting in a homogeneous solution, the probability of collisions between molecular chains is high, easily leading to inter-chain crosslinking side reactions that broaden the molecular weight distribution of the product and affect the uniformity of subsequent performance, by fixing β-CD bromide onto the surface of magnetic particles, it is more effectively separated from impurities, thereby improving the purity of stigmasterol. Detailed Implementation

[0017] To further illustrate the methods and effects of this invention, the technical solution of this invention will be clearly and completely described below in conjunction with experiments.

[0018] Example 1: A method for efficiently separating stigmasterol monomers using amphiphilic glycoside derivatives, comprising the following steps: S1, Capture Stigmasterol At 23~27℃, the amphiphilic glycoside derivative β-CD-PNIPAM-MPA was dissolved in deionized water at a ratio of 1g:68mL. Then, phytosterol was added at a mass ratio of 1:10 between the amphiphilic glycoside derivative and phytosterol. The pH was adjusted to 6.8, and the mixture was stirred for 2.3h to capture stigmasterol. In S1, the pH was adjusted by adding 0.15M phosphate buffer at a rate of 8mL / min. In S1, the preparation method of the amphiphilic glycoside derivative β-CD-PNIPAM-MPA is as follows: S1-1. At a temperature of 25℃, β-cyclodextrin was dissolved in anhydrous DMSO at a mass ratio of 1g:10mL. Nitrogen gas was introduced and the mixture was cooled to 0℃. Then, 2-bromoisobutyryl bromide was added dropwise at a molar ratio of β-cyclodextrin:2-bromoisobutyryl bromide of 1:7, with the addition time controlled at 27min. The mixture was stirred for 2.3h, centrifuged at 8000rpm for 8min to collect the precipitate, washed three times with cold diethyl ether at 3℃, and centrifuged again at 8000rpm for 8min to collect the precipitate. The precipitate was then vacuum dried at 60℃ and -0.08MPa for 6h to obtain β-CD bromide. S1-2. β-CD bromide, NIPAM, CuBr, and PMDETA were mixed in a molar ratio of Br atoms, NIPAM, CuBr, and PMDETA of 1:50:0.1:0.12 to obtain a mixture. Then, the mixture was dissolved in a 78% methanol-water solution at a ratio of 1g:8mL for β-CD bromide. Nitrogen gas was introduced at a rate of 0.18L / min for 28min, and the mixture was reacted at 35℃ for 6h. Unreacted monomers were removed by membrane dialysis, and the mixture was treated at -45℃ and 0.13mbar for 22h to obtain β-CD-PNIPAM. In S1-2, membrane dialysis purification uses a membrane with a molecular weight cutoff of 3500 Da. The purification method is as follows: the liquid to be purified is poured into the feed tank, the feed pressure is controlled at 0.3 MPa, and the discharge pressure is 0.2 MPa. When the retentate in the ceramic membrane tube cannot circulate, purified water is added. The amount of purified water added is 3 times the amount of retentate added in 4 separate additions. S1-3. According to the molar ratio of Br atom in β-CD-PNIPAM: 3-mercaptopropionic acid: PBS buffer of 1:5, and the ratio of β-CD-PNIPAM to PBS buffer of 1g:8mL, β-CD-PNIPAM and 3-mercaptopropionic acid were dissolved in PBS buffer of pH=7.4, stirred at 25℃ for 23h, purified by dialyzing, and then lyophilized to obtain an amphiphilic glycoside derivative, denoted as β-CD-PNIPAM-MPA; In S1-3, the freeze-drying process is as follows: First, the material temperature is lowered to -45℃ by vacuuming the freeze dryer. The temperature is increased by 8℃ every 2 hours. When the temperature reaches above 0℃, the temperature is increased by 7℃ every 3 hours. When the temperature shown by the upper, middle and lower temperature probes reaches 40℃ and is maintained for more than 12 hours, the material can be discharged. S2, precipitation of stigmasterol-glycoside complex The stigmasterol in S1 was heated to 45°C at a rate of 1°C / min and kept at that temperature for 22 min. It was then centrifuged at 5800 rpm for 18 min to separate the solid and liquid components and precipitate the stigmasterol-glycoside complex. S3. Obtain crude stigmasterol product The stigmasterol-glycoside complex was washed three times with a 14% ethanol aqueous solution at 45°C, and then centrifuged at 5750 rpm for 18 min to obtain a centrifuged liquid and a solid phase. The centrifuged liquid was cooled, kept warm, filtered, and dried to obtain crude stigmasterol. The solid phase was dissolved in deionized water at 25°C, filtered to remove impurities, and then freeze-dried at -50°C and 10 Pa for 6 h to recover β-CD-PNIPAM-MPA for future use. In S3, the parameters for cooling, keeping warm, filtering, and drying were as follows: natural cooling to 3°C, then keeping warm for 1.8 h, centrifuging and filtering at 5800 rpm, and drying at 90°C for 6 h. S4, refined stigmasterol The crude stigmasterol was added to 98wt% n-butanol at a ratio of 1g:3.2mL and recrystallized three times to obtain the stigmasterol monomer. In S4, the recrystallization method was as follows: stirring and heating at a rate of 8℃ / min to 64℃ and holding for 18min, then naturally cooling to 48℃ and holding for 3.7h; the stirring and heating and natural cooling steps were repeated twice.

[0019] Example 2: Unlike Example 1, at 23°C, the amphiphilic glycoside derivative β-CD-PNIPAM-MPA was dissolved in deionized water at a ratio of 1g:60mL. Then, phytosterol was added at a mass ratio of 1:8 between the amphiphilic glycoside derivative and phytosterol. The pH was adjusted to 6.5, and the mixture was stirred for 2 hours to capture stigmasterol.

[0020] Example 3: Unlike Example 1, at 27°C, the amphiphilic glycoside derivative β-CD-PNIPAM-MPA was dissolved in deionized water at a ratio of 1g:75mL. Then, phytosterol was added at a mass ratio of 1:12 between the amphiphilic glycoside derivative and phytosterol. The pH was adjusted to 7.0, and the mixture was stirred for 2.5h to capture stigmasterol.

[0021] Example 4: Unlike Example 1, the pH adjustment method is as follows: 0.1M phosphate buffer is added dropwise at a rate of 5 mL / min.

[0022] Example 5: Unlike Example 1, the pH adjustment method is as follows: 0.2M phosphate buffer is added dropwise at a rate of 10 mL / min.

[0023] Example 6: Unlike Example 1, in the preparation method of the amphiphilic glycoside derivative β-CD-PNIPAM-MPA, β-cyclodextrin was dissolved in anhydrous DMSO at a mass ratio of 1g:8mL at a temperature of 23°C. Nitrogen gas was introduced and the mixture was cooled to 0°C. Then, 2-bromoisobutyryl bromide was added dropwise at a molar ratio of 1:6 of β-cyclodextrin to 2-bromoisobutyryl bromide, with the addition time controlled at 25min. The mixture was then stirred for 2h.

[0024] Example 7: Unlike Example 1, in the preparation method of the amphiphilic glycoside derivative β-CD-PNIPAM-MPA, β-cyclodextrin was dissolved in anhydrous DMSO at a mass ratio of 1g:12mL under the condition of 23~27℃, nitrogen gas was introduced and the mixture was cooled to 0℃, and then 2-bromoisobutyryl bromide was added dropwise at a molar ratio of 1:9 of β-cyclodextrin:2-bromoisobutyryl bromide, with the addition time controlled at 30min, and then stirred for 2.5h.

[0025] Example 8: Unlike Example 1, the precipitate was collected by centrifugation at 7500 rpm for 5 min, washed twice with cold diethyl ether at 0°C, and then collected by centrifugation at 7500 rpm for 5 min. The precipitate was then vacuum dried at 55°C and -0.06 MPa for 4 h to obtain β-CD bromide.

[0026] Example 9: Unlike Example 1, the precipitate was collected by centrifugation at 8500 rpm for 10 min, washed 4 times with cold diethyl ether at 5°C, and then collected by centrifugation at 8500 rpm for 10 min. The precipitate was then vacuum dried at 65°C and -0.095 MPa for 8 h to obtain β-CD bromide.

[0027] Example 10: Unlike Example 1, β-CD bromide, NIPAM, CuBr, and PMDETA were mixed in a molar ratio of Br atoms, NIPAM, CuBr, and PMDETA of 1:45:0.1:0.12 to obtain a mixture. Then, the mixture was dissolved in a 75% methanol-water solution at a ratio of 1g:5mL for β-CD bromide. Nitrogen gas was introduced at 0.15L / min for 25min, and the reaction was carried out at 30°C for 5.5h. Unreacted monomers were removed by membrane dialysis, and the mixture was treated at -40°C and 0.1mbar for 20h to obtain β-CD-PNIPAM.

[0028] Example 11: Unlike Example 1, β-CD bromide, NIPAM, CuBr, and PMDETA were mixed in a molar ratio of Br atoms, NIPAM, CuBr, and PMDETA of 1:55:0.1:0.12 to obtain a mixture. Then, the mixture was dissolved in an 80% methanol-water solution at a ratio of 1g:10mL for β-CD bromide. Nitrogen gas was introduced at a rate of 0.2L / min for 30min, and the reaction was carried out at 40°C for 6.5h. Unreacted monomers were removed by membrane dialysis, and the mixture was treated at -50°C and 0.15mbar for 24h to obtain β-CD-PNIPAM.

[0029] Example 12: Unlike Example 1, membrane dialysis purification uses a membrane with a molecular weight cutoff of 3500 Da. The purification method is as follows: the liquid to be purified is poured into the feed tank, the feed pressure is controlled at 0.1 MPa, and the discharge pressure is 0.05 MPa. When the retentate in the ceramic membrane tube cannot circulate, purified water is added. The amount of purified water added is three times the amount of retentate added in three separate additions.

[0030] Example 13: Unlike Example 1, membrane dialysis purification uses a membrane with a molecular weight cutoff of 3500 Da. The purification method is as follows: the liquid to be purified is poured into the feed tank, the feed pressure is controlled at 0.3 MPa, and the discharge pressure is 0.2 MPa. When the retentate in the ceramic membrane tube cannot circulate, purified water is added. The amount of purified water added is 3 times the amount of retentate added in 4 separate additions.

[0031] Example 14: Unlike Example 1, β-CD-PNIPAM and 3-mercaptopropionic acid were dissolved in PBS buffer at pH 7.4 according to a molar ratio of Br atom in β-CD-PNIPAM: 3-mercaptopropionic acid: PBS buffer of 1:4 and a ratio of β-CD-PNIPAM to PBS buffer of 1g: 5mL. The mixture was stirred at 23°C for 22h, purified by dialyzing, and then lyophilized to obtain an amphiphilic glycoside derivative, denoted as β-CD-PNIPAM-MPA.

[0032] Example 15: Unlike Example 1, β-CD-PNIPAM and 3-mercaptopropionic acid were dissolved in PBS buffer at pH 7.4 according to a molar ratio of Br atom in β-CD-PNIPAM: 3-mercaptopropionic acid: PBS buffer of 1:6 and a ratio of β-CD-PNIPAM to PBS buffer of 1g:10mL. The mixture was stirred at 27°C for 24h, purified by dialyzing, and then lyophilized to obtain an amphiphilic glycoside derivative, denoted as β-CD-PNIPAM-MPA.

[0033] Example 16: Unlike Example 1, the freeze-drying process is as follows: First, the material temperature is lowered to -45°C by vacuuming with a freeze dryer. The temperature is increased by 5°C every 1 hour. When the temperature reaches above 0°C, it is increased by 5°C every 2 hours. When the temperature shown by the upper, middle and lower temperature probes reaches 40°C and is maintained for more than 12 hours, the material can be discharged.

[0034] Example 17: Unlike Example 1, the freeze-drying process is as follows: First, the material temperature is lowered to -45°C by vacuuming with a freeze dryer. The temperature is increased by 10°C every 3 hours. When the temperature reaches above 0°C, the temperature is increased by 10°C every 4 hours. When the temperature shown by the upper, middle and lower temperature probes reaches 40°C and is maintained for more than 12 hours, the material can be discharged.

[0035] Example 18: Unlike Example 1, in S2, the stigmasterol in S1 was heated to 40°C at a rate of 1°C / min, kept at that temperature for 15 min, and centrifuged at 5500 rpm for 15 min to separate the solid and liquid phases and precipitate the stigmasterol-glycoside complex.

[0036] Example 19: Unlike Example 1, in S2, the stigmasterol in S1 was heated to ℃ at a rate of 1℃ / min, kept at that temperature for 30 min, and centrifuged at 6000 rpm for 20 min to separate the solid and liquid phases and precipitate the stigmasterol-glycoside complex.

[0037] Example 20: Unlike Example 1, in S3, the stigmasterol-glycoside complex was washed twice with a 13% ethanol aqueous solution at 40°C, and then centrifuged at 5500 rpm for 15 min to obtain a centrifuged liquid and a solid phase. The centrifuged liquid was cooled, kept warm, filtered, and dried to obtain crude stigmasterol. The solid phase was dissolved in deionized water at 25°C, filtered to remove impurities, and then freeze-dried at -45°C and a vacuum of 10 Pa for 4 h to recover β-CD-PNIPAM-MPA for future use.

[0038] Example 21: Unlike Example 1, in S3, the stigmasterol-glycoside complex was washed three times with a 15% ethanol aqueous solution at 50°C, and then centrifuged at 6000 rpm for 20 min to obtain a centrifuged liquid and a solid phase. The centrifuged liquid was cooled, kept warm, filtered, and dried to obtain crude stigmasterol. The solid phase was dissolved in deionized water at 25°C, filtered to remove impurities, and then freeze-dried at -55°C and a vacuum of 10 Pa for 8 h to recover β-CD-PNIPAM-MPA for future use.

[0039] Example 22: Unlike Example 1, in S3, the parameters for cooling, heat preservation, filtration, and drying are as follows: natural cooling to 2°C, then heat preservation for 1.5 hours, centrifugal filtration at 5500 rpm, and drying at 85°C for 4 hours.

[0040] Example 23: Unlike Example 1, in S3, the parameters for cooling, heat preservation, filtration, and drying are as follows: natural cooling to 4°C, then heat preservation for 2 hours, centrifugal filtration at 6000 rpm, and drying at 95°C for 8 hours.

[0041] Example 24: Unlike Example 1, in S4, crude stigmasterol was added to 98wt% n-butanol at a ratio of 1g:2.5mL and recrystallized twice to obtain stigmasterol monomer. The recrystallization method was as follows: stirring and heating at a rate of 5℃ / min to 60℃ and holding for 15min, then naturally cooling to 45℃ and holding for 3.5h; the stirring and heating and natural cooling steps were repeated twice.

[0042] Example 25: Unlike Example 1, in S4, crude stigmasterol was added to 98wt% n-butanol at a ratio of 1g:4.0mL and recrystallized three times to obtain stigmasterol monomer. The recrystallization method was as follows: stirring and heating at a rate of 10℃ / min to 68℃ and holding for 20min, then naturally cooling to 50℃ and holding for 4h; the stirring and heating and natural cooling steps were repeated twice.

[0043] Example 26: Unlike Example 1, before S1-2, brominated β-CD was loaded onto a carboxylated support; the loading method was as follows: First, magnetic Fe3O4 nanoparticles were synthesized by coprecipitation, and then 3-aminopropyltriethoxysilane was added for amination modification to obtain amination-modified magnetic nanoparticles; the preparation method is based on existing technology. Next, the aminated magnetic nanoparticles were dissolved in anhydrous DMSO at a ratio of 1g:100mL and ultrasonically dispersed for 27min to form a suspension. The suspension was then placed in an ice bath at 3℃. A 0.4mol / L succinic anhydride solution was added at a molar ratio of 1:2.5 between the amino groups on the surface of the aminated magnetic nanoparticles and the succinic anhydride solution. The mixture was stirred for 0.7h and then stirred at 24℃ for 18h. After standing, the mixture was treated with a 0.4T neodymium iron boron permanent magnet close to the container wall for 10min. The supernatant was discarded, the solid product was collected, and washed 2-3 times with anhydrous DMSO to obtain the carboxylated support. Then, the carboxylated support, β-CD bromide, and 4-dimethylaminopyridine were dispersed in anhydrous DMSO at a mass ratio of 1:1.5:0.5:0.05. The mixture was ultrasonically stirred to form a dispersion and stirred at 24°C in the dark for 24-48 hours under nitrogen protection. Finally, the mixture was treated with a neodymium iron boron permanent magnet with a magnetic field strength of 0.4T near the container wall for 13 minutes. The supernatant was discarded, the solid product was collected, and washed sequentially with DMSO, ethanol, and deionized water. After vacuum drying at 40°C for 11 hours, β-CD bromide loaded on the carboxylated support was obtained.

[0044] Example 27: Unlike Example 26, aminated magnetic nanoparticles were dissolved in anhydrous DMSO at a ratio of 1g:200mL and ultrasonically dispersed for 25min to form a suspension. The suspension was then placed in an ice bath at 0°C. A 0.2mol / L succinic anhydride solution was added at a molar ratio of 1:2 between the amino groups on the surface of the aminated magnetic nanoparticles and the succinic anhydride solution. The mixture was stirred for 0.5h and then stirred for another 12h at 23°C. After standing, the mixture was treated with a 0.3T neodymium iron boron permanent magnet close to the container wall for 5min. The supernatant was discarded, the solid product was collected, and washed twice with anhydrous DMSO to obtain the carboxylated carrier.

[0045] Example 28: Unlike Example 26, aminated magnetic nanoparticles were dissolved in anhydrous DMSO at a ratio of 3g:200mL and ultrasonically dispersed for 30min to form a suspension. The suspension was then placed in an ice bath at 5°C. A 0.5mol / L succinic anhydride solution was added at a molar ratio of 1:3 between the amino groups on the surface of the aminated magnetic nanoparticles and the succinic anhydride solution. The mixture was stirred for 1h and then stirred for another 24h at 25°C. After standing, the mixture was treated with a 0.5T NdFeB permanent magnet near the container wall for 15min. The supernatant was discarded, the solid product was collected, and washed three times with anhydrous DMSO to obtain the carboxylated support.

[0046] Example 29: Unlike Example 26, the carboxylated support, β-CD bromide, and 4-dimethylaminopyridine were dispersed in anhydrous DMSO at a mass ratio of 1:1.2:0.4:0.04. The mixture was ultrasonically stirred to form a dispersion, and stirred at 23°C in the dark for 24 hours under nitrogen protection. Finally, the mixture was treated with a neodymium iron boron permanent magnet with a magnetic field strength of 0.3T near the container wall for 10 minutes. The supernatant was discarded, the solid product was collected, and washed sequentially with DMSO, ethanol, and deionized water. After vacuum drying at 38°C for 10 hours, β-CD bromide loaded on the carboxylated support was obtained.

[0047] Example 30: Unlike Example 26, the carboxylated support, β-CD bromide, and 4-dimethylaminopyridine were dispersed in anhydrous DMSO at a mass ratio of 1:1.7:0.6:0.06. The mixture was ultrasonically stirred to form a dispersion and stirred at 25°C in the dark for 48 hours under nitrogen protection. Finally, the mixture was treated with a neodymium iron boron permanent magnet with a magnetic field strength of 0.5 T near the container wall for 15 minutes. The supernatant was discarded, the solid product was collected, and washed sequentially with DMSO, ethanol, and deionized water. After vacuum drying at 42°C for 12 hours, β-CD bromide loaded on the carboxylated support was obtained.

[0048] Experimental Example: The description of this experimental example is based on the scheme described in Example 1, and aims to illustrate the practical application effect of the present invention.

[0049] 1. Investigate the effect of the preparation method of the amphiphilic glycoside derivative β-CD-PNIPAM-MPA on the separation effect of stigmasterol monomer: Comparative Example 1: Unlike Example 1, hydroxypropyl-γ-cyclodextrin was used instead of β-cyclodextrin in the preparation method of the amphiphilic glycoside derivative.

[0050] Comparative Example 2: Unlike Example 1, maltodextrin was used instead of β-cyclodextrin in the preparation method of the amphiphilic glycoside derivative.

[0051] Comparative Example 3: Unlike Example 1, 3-carboxyphenylboronic acid was used instead of 3-mercaptopropionic acid in the preparation method of the amphiphilic glycoside derivative.

[0052] Comparative Example 4: Unlike Example 1, the freeze-drying method was to eliminate the gradient heating and instead heat the crude stigmasterol in n-butanol until it was completely dissolved, and then let it stand at 48°C to crystallize.

[0053] Table 1. Comparison of the purity and product yield of stigmasterol monomers obtained from Examples 1, 6-17, and Comparative Examples 1-4

[0054] Conclusion: A comparison of Examples 1, 6-7, and Control Example 1 shows that if hydroxypropyl-γ-cyclodextrin is used instead of β-cyclodextrin, the cavity becomes too large, resulting in the separation of four long-chain sterols. This means that the cavity of HP-γ-CD not only encapsulates the target product stigmasterol but also almost indiscriminately encapsulates structurally similar products such as sitosterol and campesterol. During the S1 capture stage, HP-γ-CD simultaneously captures multiple sterols. In the subsequent S3 washing and S4 recrystallization, it is difficult to completely separate these structurally similar sterols. Therefore, the final product obtained... The obtained "stigmasterol monomer" is mixed with a considerable proportion of other sterols, resulting in a significant decrease in purity; and due to the lack of selectivity in the capture process, the "effective capture sites" of β-CD-PNIPAM-MPA are occupied by non-target sterols, which means that the proportion of sites used to capture target stigmasterol is reduced. At the same time, in the washing step of S3, more washing may be required to remove these unwanted sterols, which will also cause some of the captured stigmasterol to be eluted, resulting in the loss of stigmasterol and thus reducing the yield; and Control Example 1 has the problem of high cost. A comparison of Examples 1, 6-7, and Comparative Example 2 shows that in Comparative Example 2, maltodextrin was used instead of β-cyclodextrin, which prevented the formation of hydrophobic cavities. Separation could only rely on grafting groups. Due to the loss of the core separation mechanism of molecular recognition and selective inclusion of β-CD cavities, PNIPAM and MPA could only provide general hydrophobic interactions. These interactions had almost no selectivity for stigmasterol, sitosterol, etc. Therefore, the entire capture process was random, and the resulting complex was a mixture of various sterols. The purity of the final product would be very low, and it might not be possible to effectively purify it through recrystallization. Furthermore, due to the lack of strong inclusion interactions, relying solely on weak hydrophobic interactions, the "capture" efficiency would be much lower than in Example 1. The binding of stigmasterol to the material was not strong, and it was easily detached during the S2 heating precipitation and S3 washing processes, resulting in most of the stigmasterol remaining in the mother liquor and being unable to be recovered. Therefore, the yield would drop sharply.

[0055] A comparison of Examples 1, 14-15, and Comparative Example 3 shows that replacing 3-mercaptopropionic acid with 3-carboxyphenylboronic acid in Comparative Example 3 also reduces the purity and yield of the obtained stigmasterol monomer. This is because the thioester bond (-S-CO-) binding mechanism is a ligand covalent bond, and through pH control, the separation factor between stigmasterol and β-sitosterol reaches above 3.8. However, the borate ester bond binding mechanism is based on the ortho-dihydroxy group, which does not bind to sterols. The chemical structures of sterol molecules such as stigmasterol and β-sitosterol are mainly composed of inert alkane skeletons and hydroxyl groups, completely lacking the key recognition site of cis-ortho-dihydroxy. Therefore, the borate ester bond binding mechanism cannot produce any effective interaction with sterols.

[0056] A comparison of Examples 1, 16-17, and Comparative Example 4 shows that in Comparative Example 4, the lyophilization method without the gradient heating step resulted in a decrease in the purity and product yield of the obtained stigmasterol monomer. This is because the gradient heating lengthened the precipitation time difference between stigmasterol and sitosterol. By controlling the cooling rate, conditions were created for the preferential precipitation of stigmasterol, while sitosterol was retained in the mother liquor to the maximum extent, thereby greatly improving the separation purity. Furthermore, the mild nucleation conditions of the gradient heating ensured the formation of high-quality large crystals, which not only improved the purity (fewer defects) but also reduced the loss caused by excessively fine crystals passing through the filter or adhering, thus ensuring a high yield.

[0057] Based on this, the amphiphilic glycoside derivative provided in this application can play a breakthrough role in the efficient separation of stigmasterol monomers.

[0058] 2. To investigate methods for separating stigmasterol monomers and the effect of loading β-CD bromide onto a carboxylated support on the separation effect of stigmasterol monomers. Comparative Example 5: Unlike Example 1, in S4, the recrystallization method lacks the step of naturally cooling to 48°C and maintaining it for 3.7 hours.

[0059] Comparative Example 6: Unlike Example 26, the catalyst was 4-pyrrolidinylpyridine.

[0060] Table 2 Comparison of the purity and product yield of stigmasterol monomers obtained from Examples 1-5, Examples 18-30, and Comparative Examples 5-6

[0061] Conclusion: A comparison of Example 1, Example-Example, and Comparative Example 4 shows that holding at 48°C for 3.7 hours provides sufficient and stable time for crystal growth. This allows stigmasterol crystals to grow slowly and orderly, forming large crystals with complete crystal structure and higher purity. Large crystals are easier to filter, and contain fewer impurities (mother liquor and potentially co-crystallized stigmasterol). The lack of a holding step in Comparative Example 4 means that after cooling to 48°C, the system instantly reaches maximum supersaturation, explosively forming a large number of tiny crystal nuclei. This is followed immediately by another round of heating and dissolution. These microcrystals that did not have time to grow may serve as heterogeneous nucleation sites for the next round of dissolution. Tiny crystals have a huge specific surface area, making them extremely easy to adsorb and encapsulate stigmasterol impurities in the solution. Subsequent washing is difficult to remove impurities encapsulated inside the crystals, leading to a significant decrease in the purity of the final product. Furthermore, the small crystals are prone to penetrating the filter membrane or forming a dense filter cake during filtration, resulting in low washing efficiency and significant product loss, thereby reducing the yield.

[0062] A comparison of Examples 1 and 26-30 shows that loading β-CD bromide onto a carboxylated support can further improve the purity and yield of stigmasterol monomers. This is because, compared to the schemes in Examples 1-25 where β-CD-PNIPAM-MPA is dissolved in solution, relying solely on centrifugation in the S2 precipitation step may result in some small aggregates not being completely centrifuged, and the centrifugation operation is cumbersome, easily causing losses during sample transfer, leading to reduced efficiency. Examples 26-30, however, utilize the superparamagnetic property of the introduced material to achieve… Instant solid-liquid separation is a more thorough, faster, and simpler method that effectively reduces material loss and directly increases the yield of stigmasterol. Furthermore, in the homogeneous solution used in Example 1 for ATRP polymerization and MPA grafting, the high probability of molecular chain collisions and the resulting cross-linking side reactions that broaden the molecular weight distribution of the product and affect the uniformity of subsequent performance are addressed by fixing brominated β-CD on the surface of magnetic particles to obtain a more regular β-CD-PNIPAM structure, which can more effectively separate from impurities and thus improve the purity of stigmasterol.

[0063] A comparison of Examples 26, 29-30, and Comparative Example 5 shows that replacing the catalyst with 4-pyrrolidinylpyridine in Comparative Example 5 reduces the purity of the obtained stigmasterol monomer. This is because although 4-pyrrolidinylpyridine has higher reactivity and greater steric hindrance, its use may cause the connection points of brominated β-CD on the support to become random and heterogeneous. In the S1 capture stage, the material's recognition of stigmasterol depends on the synergistic interface formed by the β-CD cavity and the PNIPAM chain. The heterogeneity of the interface structure and responsiveness will cause a decrease in molecular recognition accuracy, weakening the ability to distinguish between stigmasterol and β-sitosterol (separation factor), resulting in more β-sitosterol being co-captured, thus reducing the purity of the final product. The high activity of 4-pyrrolidinylpyridine makes the loading rate comparable to or even slightly higher than that of DMAP, thereby ensuring that there are enough active sites for capture, with a smaller impact on the yield. Considering all factors, Example 26 is the optimal solution.

[0064] In summary, the method provided in this application can efficiently separate stigmasterol monomers.

Claims

1. A method for efficiently separating stigmasterin monomers using an amphiphilic glycoside derivative, characterized by, The method comprises the following steps: S1, capturing stigmasterol The amphiphilic glycoside derivative β-CD-PNIPAM-MPA is dissolved in deionized water at a proportion of 1 g: 60-75 mL at 23-27°C, then plant sterols are added at a mass ratio of 1:8-12, the pH is adjusted to 6.5-7.0, and the mixture is stirred for 2-2.5 h to capture stigmasterol; S2, precipitating stigmasterol-glycoside complex The stigmasterol obtained in S1 is warmed to 40-50°C at a rate of 1°C / min, and kept at this temperature for 15-30 min, and then centrifuged at a speed of 5500-6000 rpm for 15-20 min to separate the solid and liquid phases and precipitate the stigmasterol-glycoside complex; S3, obtaining stigmasterol crude product The stigmasterol-glycoside complex is washed with 40-50°C ethanol aqueous solution with a mass concentration of 13-15% for 2-3 times, and then centrifuged at a speed of 5500-6000 rpm for 15-20 min to obtain a centrifugate and a solid phase material, the centrifugate is cooled, kept, filtered and dried to obtain stigmasterol crude product, and the solid phase material is dissolved in deionized water at 25°C, filtered and dried under the conditions of a temperature of -45 to -55°C and a vacuum degree of ≤10 Pa for 4-8 h to recover β-CD-PNIPAM-MPA for next use; S4, stigmasterol refining The stigmasterol crude product is added into n-butanol with a concentration of 98 wt% at a proportion of 1 g: 2.5-4.0 mL for recrystallization 2-3 times to obtain stigmasterol monomer.

2. The method for high performance separation of stigmastanol monomers using amphiphilic glycoside derivatives according to claim 1, characterized in that, In S1, the preparation method of the amphiphilic glycoside derivative β-CD-PNIPAM-MPA is as follows: S1-1, β-cyclodextrin is dissolved in anhydrous DMSO at a mass ratio of 1 g: 8-12 mL at a temperature of 23-27°C, nitrogen is introduced and cooled to 0°C, then 2-bromoisobutyryl bromide is added dropwise at a molar ratio of β-cyclodextrin: 2-bromoisobutyryl bromide of 1:6-9, the dropwise time is controlled within 25-30 min, then the mixture is stirred for 2-2.5 h, centrifuged at a speed of 7500-8500 rpm for 5-10 min to collect the precipitate, washed with cold ether at 0-5°C for 2-4 times, and then centrifuged at a speed of 7500-8500 rpm for 5-10 min to collect the precipitate, which is vacuum dried at a temperature of 55-65°C and a pressure of -0.06 to -0.095 MPa for 4-8 h to obtain brominated β-CD; S1-2, the bromide β-CD, NIPAM, CuBr and PMDETA are mixed according to the molar ratio of Br atoms in bromide β-CD, NIPAM, CuBr and PMDETA is 1:45-55:0.1:0.12 to obtain a mixture, then the mixture is dissolved in a methanol-water solution with a mass fraction of 75-80% according to the ratio of bromide β-CD to methanol-water solution is 1g:5-10mL, nitrogen is introduced at a flow rate of 0.15-0.2L / min for 25-30min, then the reaction is carried out at 30-40℃ for 5.5-6.5h, the unreacted monomers are removed by membrane dialysis purification, and the β-CD-PNIPAM is obtained by treating at-40--50℃, 0.1-0.15mbar for 20-24h; S1-3, the β-CD-PNIPAM and 3-mercaptopropionic acid are dissolved in the PBS buffer solution with pH=7.4 according to the molar ratio of Br atoms in β-CD-PNIPAM:3-mercaptopropionic acid is 1:4-6, and the ratio of β-CD-PNIPAM to PBS buffer solution is 1g:5-10mL, stirring at 23-27℃ for 22-24h, freeze-drying after dialysis purification, the amphiphilic glycoside derivative is obtained, denoted as β-CD-PNIPAM-MPA.

3. The method for high performance separation of stigmastanol monomers using amphiphilic glycoside derivatives according to claim 1, characterized in that, In S1, the pH adjusting method is: dropping with a concentration of 0.1-0.2M phosphate buffer solution at a dropping rate of 5-10mL / min.

4. The method for high performance separation of stigmastanol monomers using amphiphilic glycoside derivatives according to claim 1, characterized in that, In S3, the parameters of cooling, incubation, filtration and drying are: natural cooling to 2-4℃, then incubation for 1.5-2h, centrifugal filtration at a rotation speed of 5500-6000rpm, and drying at a temperature of 85-95℃ for 4-8h.

5. The method for high performance separation of stigmastanol monomers using amphiphilic glycoside derivatives according to claim 1, wherein, In S4, the recrystallization method is: stirring and heating to 60-68℃ at a heating rate of 5-10℃ / min and maintaining for 15-20min, natural cooling to 45-50℃ and maintaining for 3.5-4h; repeating the stirring and heating, and natural cooling steps for 1-2 times.

6. The method for high performance separation of stigmastanol monomers using amphiphilic glycoside derivatives according to claim 2, wherein, In S1-3, the freeze-drying process is: first, the material temperature is reduced to-45℃ after vacuumizing with the freeze dryer, then the temperature is increased by 5-10℃ every 1-3h, when the temperature reaches above 0℃, the temperature is increased by 5-10℃ every 2-4h, when the temperature of the upper, middle and lower temperature probes reaches 40℃ and maintains for 12h or more, the material can be discharged.

7. The method for high performance separation of stigmastanol monomers using amphiphilic glycoside derivatives according to claim 1, wherein, In S1-2, the membrane dialysis purification adopts a membrane with a molecular weight cut-off of 3500Da, and the purification method is: the material liquid to be purified is poured into a feeding barrel, the feeding pressure is controlled to be ≤0.3MPa, the discharging pressure is controlled to be ≤0.2MPa, when the circulation of the cut-off liquid in the ceramic membrane tube is impossible, the purified water is supplemented, and the amount of the purified water added is 3 times the amount of the cut-off liquid. ​ 8. The method for high performance separation of stigmastanol monomers using amphiphilic glycoside derivatives according to claim 1, wherein, Before S1-2, the bromide β-CD is loaded on a carboxylated carrier; the loading method is: First, the magnetic nano Fe3O4 particles are synthesized by co-precipitation method, and 3-aminopropyltriethoxysilane is added for amination modification treatment to obtain aminated magnetic nanoparticles; Secondly, the amino-functionalized magnetic nanoparticles were dissolved in anhydrous DMSO in a ratio of 1-3 g:200 mL, ultrasonic dispersion for 25-30 min to form a suspension, then the suspension was placed in an ice bath at 0-5℃, a succinic anhydride solution with a concentration of 0.2-0.5 mol / L was added according to the molar ratio of the amino groups on the surface of the amino-functionalized magnetic nanoparticles to the succinic anhydride solution of 1:2-3, stirring for 0.5-1 h, then continuing to stir at 23-25℃ for 12-24 h, standing, and treating with a 0.3-0.5 T neodymium-iron-boron permanent magnet close to the container wall for 5-15 min, pouring off the supernatant, collecting the solid product, and washing with anhydrous DMSO for 2-3 times to obtain the carboxyl-functionalized carrier; Then, the carboxyl-functionalized carrier, brominated β-CD and catalyst were dispersed in anhydrous DMSO in a mass ratio of 1:1.2-1.7:0.4-0.6:0.04-0.06, ultrasonic stirring to form a dispersion, continuing to stir at 23-25℃ under the protection of nitrogen for 24-48 h in the dark, finally treating with a 0.3-0.5 T neodymium-iron-boron permanent magnet close to the container wall for 10-15 min, pouring off the supernatant, collecting the solid product, and washing with DMSO, ethanol and deionized water in turn, and vacuum drying at 38-42℃ for 10-12 h to obtain the brominated β-CD loaded on the carboxyl-functionalized carrier.

9. The method for high performance separation of stigmastanol monomers using amphiphilic glycoside derivatives according to claim 8, characterized by, The catalyst is 4-dimethylamino pyridine.

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