A method for isolating high purity stigmasterol from mixed plant sterols

Abstract

This invention provides a method for separating high-purity stigmasterol from mixed phytosterols, comprising: (1) fractionally extracting a raw material solution containing mixed phytosterols using an extractant containing an ionic liquid to obtain an extract containing stigmasterol; wherein the mixed phytosterols include stigmasterol and at least one selected from β-sitosterol, campesterol, and brassosterol, and the ionic liquid is composed of choline cations and anions, the anions including amino acid anions and / or carboxylic acid anions; (2) back-extracting the extract containing stigmasterol, and obtaining high-purity stigmasterol after concentration and cooling crystallization of the back-extracted phase. The method of this invention has the characteristics of high separation efficiency, high product purity, low energy consumption, and environmental friendliness in separating stigmasterol from mixed phytosterols, and is suitable for industrial production.

Description

Technical Field

[0001] This invention belongs to the field of chemical separation technology, specifically relating to a method for separating high-purity stigmasterol from a mixture of phytosterols. Background Technology

[0002] Stigmasterol is a widely distributed phytosterol in the plant kingdom, attracting widespread attention due to its unique structural features and diverse physiological activities. The basic skeleton of stigmasterol consists of a cyclopentane-polyhydrophenanthrene (sterol core) and a side chain. Its most prominent structural feature is the double bond between C-22 and C-23 on the side chain, along with an ethyl group attached at C-24. The double bond and ethyl group are key structural features that distinguish it from other common phytosterols (such as β-sitosterol and campesterol). Stigmasterol is a typical representative of phytosterols, widely found in various plants and deodorized distillates from refined plant oils. It possesses various physiological activities, such as lowering serum total cholesterol and low-density lipoprotein cholesterol, scavenging free radicals, helping to reduce oxidative stress damage to cells, and inhibiting proliferation and inducing apoptosis in various cancer cell lines.

[0003] Natural plant sterols often exist as mixtures of various structurally similar sterols, with β-sitosterol, stigmasterol, campesterol, and brassosterol being the main components. These sterols share a common structural feature: their steroid nucleus. The differences are limited to the number and position of double bonds and methyl / ethyl branches on their side chains. This high structural similarity leads to very similar physicochemical properties, making the efficient enrichment of single sterol components from mixtures extremely difficult. Therefore, developing and applying a purification technique with high yield and good separation efficiency is crucial.

[0004] β-sitosterol Stigmasterol campesterol rapeseed sterol Currently, commonly used methods for purifying phytosterols include enzyme-assisted, microwave-assisted, or ultrasound-assisted solvent extraction, and recrystallization. Almeida et al. [Cláudia F. Almeida, Yaidelin A. Manrique, José Carlos B. Lopes. Recovery of ergosterol from Agaricus bisporus mushrooms via supercritical fluid extraction: A response surface methodology optimization[J], Heliyon, 2024, 10: 2] optimized the purification process of ergosterol extracted from mushrooms using supercritical CO2 extraction, achieving a purity of 54.7% with a yield of only 62%. Srividya et al. [Srividya N, Heidorn DB, Lange BM. Rapid purification of gram quantities of beta-sitosterol from acommercial phytosterol mixture[J]. BMC research notes, 2014, 7: 182] used chromatographic separation, passing the phytosterol mixture through silica gel-packed column chromatography and Na-Y molecular sieve column chromatography, achieving a 92% purity of β-sitosterol in the product received within 72 hours. Although chromatography can yield sterol products with high purity, it has a small throughput. Summary of the Invention

[0005] To address at least one of the aforementioned technical problems in the prior art, this invention provides a method for separating high-purity stigmasterol from a mixture of phytosterols using an extraction-crystallization coupling process. The method first uses an extractant containing an ionic liquid to obtain an extract rich in stigmasterol. The extract is then back-extracted and concentrated, followed by a cooling crystallization process to obtain high-purity stigmasterol and a mother liquor containing most of the stigmasterol. The crystallization step allows the extraction process to achieve high-purity stigmasterol at a lower flow ratio and fewer extraction stages. Furthermore, the mother liquor can be reused, either directly or after solvent replacement via evaporation, combined with the feed solution.

[0006] The technical solution of the present invention is as follows: (1) The raw material liquid containing mixed phytosterols is fractionally extracted with an extractant containing ionic liquid to obtain an extract containing stigmasterol; wherein the mixed phytosterols include stigmasterol and at least one selected from β-sitosterol, campesterol, and rapeseed sterol, and the ionic liquid is composed of choline cations and anions, and the anions include amino acid anions and / or carboxylic acid anions; (2) The extract containing stigmasterol is back-extracted, and the back-extracted phase is concentrated and cooled to crystallize to obtain high-purity stigmasterol.

[0007] According to some embodiments of the present invention, in the ionic liquid, the choline cation includes a choline cation or a choline cation substituted with one or more substituents selected from alkyl, alkenyl, cyano, thiocyano, sulfone, and carbonyl. In some embodiments, the alkyl group is a C1-C4 straight-chain or branched alkyl group, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, etc. In some embodiments, the alkyl group is a C2-C4 straight-chain alkyl group. In some embodiments, the alkenyl group is a C2-C4 alkenyl group, such as vinyl, propenyl, butenyl, etc.

[0008] In some embodiments, the carboxylic acid anion is a C2-C20 carboxylic acid anion. In some embodiments, the carboxylic acid anion is a C8-C20 carboxylic acid anion. In this invention, the carboxylic acid anion includes, but is not limited to, one or more of the following: octanoic acid anion, decanoic acid anion, undecanoic acid anion, dodecanoic acid anion, myristic acid anion, palmitic acid anion, lauric acid anion, stearic acid anion, and oleic acid anion. In some embodiments, the carboxylic acid anion is one or more of the following: decanoic acid anion, myristic acid anion, and palmitic acid anion.

[0009] In some embodiments, the amino acid anion is selected from one or more of glycine anion, proline anion, lysine anion, alanine anion, leucine anion, valine anion, and phenylalanine anion.

[0010] In some embodiments, the amino acid anion is glycine or lysine.

[0011] In some embodiments, the amino acid anion is replaced by one or more substituents selected from hydroxyl, C1-C4 straight-chain or branched alkyl, and C1-C4 straight-chain or branched alkenyl.

[0012] In some embodiments, the extractant is an ionic liquid.

[0013] In some embodiments, the extractant is a mixture of an ionic liquid and a polar solvent.

[0014] In some embodiments, the molar percentage of the ionic liquid in the mixture of the ionic liquid and the polar solvent is 0.01% to 50%, for example, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.5%, 0.8%, 1%, 2%, 5%, 8%, 10%, 12%, 15%, 18%, 20%, 22%, 25%, 28%, 30%, 32%, 35%, 38%, 40%, 42%, 45%, 48%, 50%.

[0015] In some embodiments, the molar percentage of the ionic liquid in the mixture of the ionic liquid and the polar solvent is 0.05% to 40%. In some embodiments, the molar percentage of the ionic liquid in the mixture of the ionic liquid and the polar solvent is 0.1% to 30.0%. In some embodiments, the molar percentage of the ionic liquid in the mixture of the ionic liquid and the polar solvent is 0.2% to 15.0%. In some embodiments, the molar percentage of the ionic liquid in the mixture of the ionic liquid and the polar solvent is 0.3% to 10.0%.

[0016] In some embodiments, the polar solvent in the mixture of the ionic liquid and the polar solvent is selected from one or more of water, oxygen-containing organic solvents, phosphorus-containing organic solvents, nitrogen-containing organic solvents, and sulfur-containing organic solvents. In some embodiments, the polar solvent is selected from one or more of water, alcohols, nitriles, amides, sulfones, and sulfoxides. In some specific embodiments, the polar solvent is selected from at least one of water, methanol, ethylene glycol, 1,3-propanediol, glycerol, dimethyl sulfoxide, sulfolane, acetonitrile, N-methylpyrrolidone, N-methylformamide, and N,N-dimethylformamide. In some embodiments, the polar solvent is selected from acetonitrile and dimethyl sulfoxide. In some preferred embodiments, the polar solvent is dimethyl sulfoxide.

[0017] According to some embodiments of the present invention, the ionic liquid is selected from one or more of choline decanoate, choline myristate, choline palmitate, choline glycidate, and choline lysine.

[0018] According to some embodiments of the present invention, the extractant is selected from one or more of the following: choline decanoate, choline myristate, choline palmitate, choline glycate, choline lysine, a mixture of choline decanoate and dimethyl sulfoxide, a mixture of choline myristate and dimethyl sulfoxide, a mixture of choline palmitate and dimethyl sulfoxide, a mixture of choline glycate and dimethyl sulfoxide, a mixture of choline lysine and dimethyl sulfoxide, and a mixture of choline decanoate and acetonitrile.

[0019] According to some embodiments of the present invention, the raw material liquid further contains a raw material solvent, which includes a nonpolar organic solvent and optionally a polarity modifier. In some embodiments, the nonpolar organic solvent includes one or more of C6-C12 straight-chain alkanes, C6-C12 branched alkanes, C6-C12 cycloalkanes, and C1-C6 haloalkanes. In some specific embodiments, the nonpolar organic solvent includes one or more of n-hexane, n-heptane, n-octane, n-nonane, n-decane, 2,2,4-trimethylpentane, isooctane, isodecane, cyclohexane, methylcyclohexane, and chloroform. In some embodiments, the nonpolar organic solvent is n-hexane.

[0020] In this invention, adding a polarity modifier to the raw material solvent can increase the solubility of the mixed phytosterol raw materials. According to some embodiments of the invention, the polarity modifier includes alcoholic organic solvents. In some embodiments, the polarity modifier includes C1-C4 monohydric alcohols and / or C2-C4 polyhydric alcohols. In some specific embodiments, the polarity modifier includes one or more of methanol, ethanol, ethylene glycol, 1,3-propanediol, and glycerol. In some embodiments, the polarity modifier is methanol.

[0021] According to some embodiments of the present invention, based on the total volume of the nonpolar organic solvent and the polar modifier being 100%, the volume percentage of the polar modifier is 0.1% to 10.0%, for example, 0.1%, 0.2%, 0.5%, 0.8%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, 8.5%, 9.0%, 9.5%, 10.0%, or any value between them, preferably 0.5% to 5.0%.

[0022] According to some embodiments of the present invention, the mixed phytosterols include stigmasterol and at least one selected from β-sitosterol, campesterol, and brassosterol, wherein the content of stigmasterol is 10 wt% to 40 wt%, for example, 10 wt%, 12 wt%, 15 wt%, 20 wt%, 25 wt%, 30 wt%, 35 wt%, 40 wt%, or any value between therewith. In some embodiments, the content of stigmasterol in the mixed phytosterols is 20 wt% to 30 wt%.

[0023] According to some embodiments of the present invention, the total concentration of phytosterols in the raw material solution is 1.0 g / L to 100.0 g / L, for example, 1.0 g / L, 2.0 g / L, 3.0 g / L, 5.0 g / L, 8 g / L, 10.0 g / L, 12.0 g / L, 15.0 g / L, 18.0 g / L, 20.0 g / L, 25.0 g / L, 30.0 g / L, 35.0 g / L, 40.0 g / L, 45.0 g / L, 50.0 g / L, 55.0 g / L, 60.0 g / L, 65.0 g / L, 70.0 g / L, 75.0 g / L, 80.0 g / L, 85.0 g / L, 90.0 g / L, 95.0 g / L, 100.0 g / L, or any value between them, preferably 5.0 g / L. g / L~50.0 g / L.

[0024] According to some embodiments of the present invention, the mixed phytosterols include stigmasterol, β-sitosterol, campesterol, and brassosterol.

[0025] According to some embodiments of the present invention, the mass ratio of β-sitosterol to stigmasterol in the mixed phytosterols is (1.0~2.0):1. According to some embodiments of the present invention, the mass ratio of campesterol to stigmasterol in the mixed phytosterols is (0.5~1.5):1. According to some embodiments of the present invention, the mass ratio of campesterol to stigmasterol in the mixed phytosterols is (0.005~0.02):1.

[0026] According to some embodiments of the present invention, the fractional extraction includes an extraction section and a washing section. The extraction section can be 5 to 12 stages, for example, 5, 6, 7, 8, 9, 10, 11, 12 stages, etc. In some embodiments, the extraction section has 5 to 10 stages. In some embodiments, the extraction section has 8 to 10 stages. The washing section can be 5 to 20 stages, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 stages, etc. In some embodiments, the washing section has 10 to 20 stages. In some embodiments, the washing section has 10 to 15 stages.

[0027] According to some embodiments of the present invention, in the fractional extraction, the flow ratio of the extractant to the feed liquid is (0.5-5):1, for example, 0.5:1, 0.8:1, 1.0:1, 1.2:1, 1.5:1, 1.8:1, 2.0:1, 2.2:1, 2.5:1, 2.8:1, 3.0:1, 3.2:1, 3.5:1, 3.8:1, 4.0:1, 4.2:1, 4.5:1, 4.8:1, 5.0:1, etc. In some embodiments, in the fractional extraction, the flow ratio of the extractant to the feed liquid is (0.5-2):1. In some embodiments, the flow ratio of extractant to feed liquid in the fractional extraction is (0.8–2):1. In some embodiments, the flow ratio of extractant to feed liquid in the fractional extraction is (0.8–1.5):1. In some embodiments, the flow ratio of extractant to feed liquid in the fractional extraction is (0.8–1.2):1.

[0028] According to some embodiments of the present invention, in the fractional extraction, the flow ratio of detergent to feed liquid is (0.1 to 10.0):1, for example, 0.1:1, 0.5:1, 0.8:1, 1.0:1, 1.2:1, 1.5:1, 1.8:1, 2.0:1, 3.0:1, 4.0:1, 5.0:1, 6.0:1, 7.0:1, 8.0:1, 9.0:1, 10.0:1, etc. In some embodiments, the flow ratio of detergent to feed liquid in the fractional extraction is (0.5 to 8.0):1. In some embodiments, the flow ratio of detergent to feed liquid in the fractional extraction is (7.0 to 8.0):1.

[0029] In this invention, unless otherwise specified, "flow ratio" refers to "volume ratio".

[0030] According to some embodiments of the present invention, the detergent is of the same type as the raw material solvent in the raw material liquid.

[0031] According to some embodiments of the present invention, the operating temperature of the fractional extraction is 10–70 °C, for example, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 40 °C, 50 °C, 60 °C, 70 °C, or any value between them. If the extraction temperature is too low, the solution viscosity is high, which is detrimental to mixing and mass transfer; if the extraction temperature is too high, the organic solvent will begin to evaporate, and the solubility of the two solvents will increase, which is also detrimental to extraction. In some preferred embodiments, the operating temperature of the fractional extraction is 20 °C–50 °C.

[0032] According to some embodiments of the present invention, in step (2), the concentration is vacuum concentration.

[0033] According to some embodiments of the present invention, in step (2), the back-extraction solvent used is a polar or non-polar organic solvent. The polar solvent includes one or more of alcohols, ketones, and esters. The non-polar organic solvent includes one or more of C6-C12 straight-chain alkanes, C6-C12 branched alkanes, C6-C12 cycloalkanes, and C1-C6 haloalkanes. In some embodiments, the back-extraction solvent includes one or more of C6-C12 straight-chain alkanes, C6-C12 branched alkanes, C6-C12 cycloalkanes, and C1-C6 haloalkanes. In some embodiments, the back-extraction solvent includes one or more of n-hexane, n-heptane, n-octane, n-nonane, n-decane, 2,2,4-trimethylpentane, isooctane, isodecane, cyclohexane, methylcyclohexane, trichloromethane, and 1,2-dichloroethane. In some embodiments, the back-extraction solvent is n-hexane or 1,2-dichloroethane.

[0034] According to some embodiments of the present invention, the cooling crystallization includes cooling the concentrated solution from 40–60 °C (e.g., 40 °C, 45 °C, 50 °C, 55 °C, 60 °C or any value between them) to -20–30 °C (e.g., -20 °C, -15 °C, -10 °C, -5 °C, 0 °C, 5 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, etc.). In some preferred embodiments, the initial temperature of the crystallization operation is 45–50 °C. According to some embodiments of the present invention, the concentrated solution is cooled to -20–10 °C. According to some embodiments of the present invention, the concentrated solution is cooled to -10–0 °C.

[0035] According to some embodiments of the present invention, in step (2), the crystallization time is 10 to 60 min, for example, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min or any value between them. In some preferred embodiments, the crystallization time is 20 to 30 min.

[0036] According to some embodiments of the present invention, the method further includes the following steps: The mother liquor obtained after crystallization is concentrated under vacuum and then returned to the raw material liquid for reuse; or, the mother liquor obtained after crystallization is concentrated under vacuum and dissolved in the raw material solvent and then returned to the raw material liquid for reuse.

[0037] Compared with the prior art, the present invention has the following beneficial technical effects: 1. This invention uses an extraction-crystallization coupling method to extract stigmasterol from mixed phytosterols. This method reduces the number of extraction stages required by using extraction methods alone, reduces the amount of extractant used in the extraction process, and overcomes the drawback of not being able to obtain high-purity products by using extraction operations alone. Compared with crystallization operations alone, this invention pre-extracts the mixed phytosterols in the raw materials using an extraction method, which increases the stigmasterol content, improves crystallization efficiency, and reduces the high energy consumption caused by using cooling crystallization processes alone, thus achieving a balance between product purity, separation efficiency, and economic benefits.

[0038] 2. The ionic liquid in the extractant used in this invention, compared with conventional organic solvents and other ionic liquids, not only has a high selective separation ability for stigmasterol, but also has good degradability and biocompatibility, reducing the impact on human health and the environment, and has broad prospects for green applications.

[0039] 3. The method of the present invention has high separation efficiency and the purity of the separated stigmasterol product is high, reaching over 95%. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0041] Unless otherwise specified, all reagents used in the following experiments of this invention are commercially available products or reagents prepared according to conventional methods. Unless otherwise specified, all methods used in the experiments are conventional experimental methods. Unless otherwise specified, all instruments used in the experiments are commercially available.

[0042] Ionic liquids, molten salts composed of cations and anions that are liquid at or near room temperature, can have their polarity, hydrophobicity, solubility, and functionality precisely controlled by altering the combination of cations and anions. They are also known as "designable solvents" and exhibit enormous application potential in separation processes due to their unique physicochemical properties. However, some commonly used ionic liquids are non-biodegradable and toxic to the environment and ecosystems. Furthermore, they are frequently used in the extraction of natural products and ultimately as food additives for humans or animals; therefore, their biological toxicity cannot be ignored.

[0043] Based on this, this application achieves the extraction of stigmasterol by selecting a type of separation medium that is highly efficient, low in toxicity, and has good biocompatibility.

[0044] As a specific embodiment of the present invention, the method for separating high-purity stigmasterol from a mixture of phytosterols includes the following steps: (S1) Dissolve the mixed phytosterol raw materials in a raw material solvent in a certain proportion to obtain a raw material solution; (S2) The feed liquid is subjected to multi-stage fractional extraction using an extractant containing ionic liquid. The extractant enters the fractional extraction system from the first stage of the extraction section, and the feed liquid enters the fractional extraction system from the last stage of the extraction section. The detergent of the same type as the feed solvent enters the fractional extraction system from the first stage of the washing section. It merges with the feed liquid in the last stage of the extraction section and enters the extraction section. After multi-stage countercurrent contact, the extract flows out from the first stage of the washing section, and the raffinate flows out from the first stage of the extraction section. The extract is collected and back-extracted and concentrated to obtain a crystalline feed liquid containing a high concentration of stigmasterol. (S3) The raw material liquid is cooled and crystallized to obtain high-purity stigmasterol and crystallization mother liquor; the crystallization mother liquor is concentrated under vacuum and then added to the raw material liquid with the same solvent.

[0045] In the following examples and comparative examples of the present invention, the concentration of each phytosterol component was analyzed by high performance liquid chromatography (HPLC). The specific HPLC analysis conditions were as follows: Waters Sunfire C18 column (4.6 mm ID×250 mm, particle size 5 μm), mobile phase was pure methanol, flow rate was 1 mL / min, column temperature was 30 °C, and UV detection wavelength was 210 nm.

[0046] In the following examples and comparative examples, the yield and purity of stigmasterol were calculated using the following methods: Stigmasterol yield = (mass of stigmasterol in product / mass of stigmasterol in raw material) × 100%; Stigmasterol purity = mass of stigmasterol in the product / total mass of the product × 100%.

[0047] The mixed phytosterol raw materials used in the following examples and comparative examples of the present invention contain β-sitosterol, stigmasterol, campesterol and brassosterol, wherein the mass ratio of β-sitosterol to stigmasterol is approximately 1.5:1, the mass ratio of campesterol to stigmasterol is approximately 1:1, and the mass ratio of brassosterol to stigmasterol is approximately 0.01:1.

[0048] Example 1

[0049] A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline decanoate-dimethyl sulfoxide (choline decanoate molar fraction of 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0050] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0051] The purity of stigmasterol in the obtained product was 95.44 wt%, and the yield was 73.02 wt%.

[0052] Example 2 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline-myristate-dimethyl sulfoxide (choline-myristate molar fraction 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0053] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0054] The purity of stigmasterol in the obtained product was 94.13 wt%, and the yield was 72.19 wt%.

[0055] Example 3 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline palmitate-dimethyl sulfoxide (choline palmitate molar fraction 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0056] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0057] The purity of stigmasterol in the obtained product was 93.25 wt%, and the yield was 72.01 wt%.

[0058] Example 4 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline glycinate-dimethyl sulfoxide (choline glycinate molar fraction 10 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0059] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0060] The purity of stigmasterol in the obtained product was 93.13 wt%, and the yield was 72.98 wt%.

[0061] Example 5 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline lysine salt-dimethyl sulfoxide (choline lysine salt molar fraction of 10 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0062] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0063] The purity of stigmasterol in the obtained product was 92.89 wt%, and the yield was 72.61 wt%.

[0064] Example 6 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline decanoate-dimethyl sulfoxide (choline decanoate molar fraction 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:8:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0065] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0066] The purity of stigmasterol in the obtained product was 92.14 wt%, and the yield was 75.21 wt%.

[0067] Example 7 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline decanoate-dimethyl sulfoxide (choline decanoate molar fraction 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 8 stages in the extraction section and 10 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0068] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0069] The purity of stigmasterol in the obtained product was 93.27 wt%, and the yield was 72.75 wt%.

[0070] Example 8 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline decanoate-dimethyl sulfoxide (choline decanoate molar fraction 3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0071] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0072] The purity of stigmasterol in the obtained product was 96.03 wt%, and the yield was 73.10 wt%.

[0073] Example 9 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 10.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline decanoate-dimethyl sulfoxide (choline decanoate molar fraction 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0074] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0075] The purity of stigmasterol in the obtained product was 94.85 wt%, and the yield was 72.56 wt%.

[0076] Example 10 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30°C using choline decanoate-acetonitrile (choline decanoate molar fraction 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0077] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0078] The purity of stigmasterol in the obtained product was 90.13 wt%, and the yield was 71.36 wt%.

[0079] Example 11 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 50 °C using choline decanoate-dimethyl sulfoxide (choline decanoate molar fraction 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0080] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0081] The purity of stigmasterol in the obtained product was 91.68 wt%, and the yield was 71.99 wt%.

[0082] Example 12 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline decanoate-dimethyl sulfoxide (choline decanoate molar fraction of 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0083] The extract was back-extracted with hexane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 30 min). The crystals were filtered to obtain high-purity stigmasterol crystals, which were then washed and dried to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed solution, which was then recovered into the feed solution.

[0084] The purity of stigmasterol in the obtained product was 95.16 wt%, and the yield was 75.23 wt%.

[0085] Example 13 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 50 °C using choline decanoate-dimethyl sulfoxide (choline decanoate molar fraction 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0086] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 60 °C and then slowly cooled to -10 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0087] The purity of stigmasterol in the obtained product was 95.30 wt%, and the yield was 72.89 wt%.

[0088] Example 14 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline decanoate-dimethyl sulfoxide (choline decanoate molar fraction of 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0089] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to 0 °C and maintained for 30 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0090] The purity of stigmasterol in the obtained product was 96.10 wt%, and the yield was 73.29 wt%.

[0091] Example 15 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline decanoate-dimethyl sulfoxide (choline decanoate molar fraction of 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0092] The extract was back-extracted with 1,2-dichloroethane, concentrated under vacuum, and then subjected to cooling crystallization (heated to 45 °C and then slowly cooled to -10 °C and maintained for 60 min). High-purity stigmasterol crystals were obtained by filtration, washing, and drying to obtain the stigmasterol product. The crystallization mother liquor was concentrated under vacuum and then mixed with n-hexane:methanol = 98:2 (v / v) to prepare a solution with the same concentration as the feed liquor, which was then recovered into the feed liquor.

[0093] The purity of stigmasterol in the obtained product was 95.91 wt%, and the yield was 73.14 wt%.

[0094] Comparative Example 1 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline decanoate-dimethyl sulfoxide (choline decanoate molar fraction 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 19.6 : 3.1 : 1, with 28 stages in the extraction section and 40 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0095] The extract was back-extracted using hexane:methanol = 98:2 (v / v), and the back-extracted phase was concentrated under vacuum to obtain stigmasterol product with a purity of 91.44 wt.%.

[0096] Comparative Example 2 A mixed phytosterol feedstock (containing 26.0 wt.% stigmasterol) was dissolved in a feedstock solvent prepared with hexane:methanol = 98:2 (v / v) to prepare a 4.0 g / L solution as the feedstock liquid. Fractional extraction was performed at 30 °C using choline decanoate-dimethyl sulfoxide (choline decanoate molar fraction of 0.3 mol%) as the extractant and hexane:methanol = 98:2 (v / v) as the washing agent. The flow ratio of extractant, washing agent, and feedstock liquid was 1:7:1, with 10 stages in the extraction section and 15 stages in the washing section. The extractant entered the fractional extraction system from the first stage of the extraction section, the feedstock liquid entered from the last stage of the extraction section, and the washing agent entered from the first stage of the washing section. A stigmasterol-rich extract flowed out from the first stage of the washing section, and a raffinate rich in other components flowed out from the first stage of the extraction section. Both the extract and the raffinate were collected.

[0097] The extract was back-extracted using hexane:methanol = 98:2 (v / v), and the back-extracted phase was concentrated under vacuum to obtain stigmasterol product with a purity of 51.29 wt.%.

[0098] The above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of this application.

Claims

1. A method for separating high-purity stigmasterol from a mixture of phytosterols, comprising the following steps: (1) The raw material solution containing mixed phytosterols was fractionally extracted using an extractant containing ionic liquid to obtain an extract containing stigmasterol; wherein, The mixed phytosterols include stigmasterol and at least one selected from β-sitosterol, campesterol, and brassosterol; the ionic liquid is composed of choline cations and anions, and the anions include amino acid anions and / or carboxylic acid anions. (2) The extract containing stigmasterol is back-extracted, and the back-extracted phase is concentrated and cooled to crystallize to obtain high-purity stigmasterol.

2. The method according to claim 1, characterized in that, The choline cations include choline cations or choline cations substituted with one or more substituents selected from alkyl, alkenyl, cyano, thiocyano, sulfone, and carbonyl; the alkyl group is preferably a C1-C4 straight-chain or branched alkyl group, more preferably a C2-C4 straight-chain alkyl group, and the alkenyl group is preferably a C2-C4 alkenyl group; and / or The carboxylic acid anion is a C2-C20 carboxylic acid anion, preferably a C8-C20 carboxylic acid anion, more preferably one or more of the following: octanoic acid anion, decanoic acid anion, undecanoic acid anion, dodecanoic acid anion, myristic acid anion, palmitic acid anion, lauric acid anion, stearic acid anion, and oleic acid anion; further preferably one or more of the following: decanoic acid anion, myristic acid anion, and palmitic acid anion; and / or The amino acid anion is selected from one or more of glycine anion, proline anion, lysine anion, alanine anion, leucine anion, valine anion, and phenylalanine anion, preferably glycine or lysine; Preferably, the ionic liquid is selected from at least one of choline decanoate, choline laurate, choline myristate, choline stearate, choline palmitate, choline glycinate, and choline lysine.

3. The method according to claim 1 or 2, characterized in that, The extractant is an ionic liquid or a mixture of an ionic liquid and a polar solvent; Preferably, in the mixture of ionic liquid and polar solvent, the molar percentage of ionic liquid is 0.01% to 50%, more preferably 0.05% to 40%, more preferably 0.1% to 30%, and even more preferably 0.2% to 15%. Preferably, in the mixture of the ionic liquid and the polar solvent, the polar solvent is selected from one or more of water, oxygen-containing organic solvents, phosphorus-containing organic solvents, nitrogen-containing organic solvents, and sulfur-containing organic solvents; preferably, the polar solvent is selected from one or more of water, alcohols, nitriles, amides, sulfones, and sulfoxides; more preferably, the polar solvent is selected from at least one of water, methanol, ethylene glycol, 1,3-propanediol, glycerol, dimethyl sulfoxide, sulfolane, acetonitrile, N-methylpyrrolidone, N-methylformamide, and N,N-dimethylformamide; even more preferably, the polar solvent is dimethyl sulfoxide or acetonitrile.

4. The method according to any one of claims 1-3, characterized in that, The extractant is selected from one or more of the following: choline decanoate, choline myristate, choline palmitate, choline lysine, choline lysine, a mixture of choline decanoate and dimethyl sulfoxide, a mixture of choline myristate and dimethyl sulfoxide, a mixture of choline palmitate and dimethyl sulfoxide, a mixture of choline glysine and dimethyl sulfoxide, a mixture of choline lysine and dimethyl sulfoxide, and a mixture of choline decanoate and acetonitrile.

5. The method according to any one of claims 1-4, characterized in that, The raw material liquid contains a raw material solvent, which includes a non-polar organic solvent and an optional polarity modifier; Preferably, the nonpolar organic solvent includes one or more of C6-C12 straight-chain alkanes, C6-C12 branched alkanes, C6-C12 cycloalkanes, and C1-C6 haloalkanes; more preferably, the nonpolar organic solvent includes one or more of n-hexane, n-heptane, n-octane, n-nonane, n-decane, 2,2,4-trimethylpentane, isooctane, isodecane, cyclohexane, methylcyclohexane, and chloroform; even more preferably, the nonpolar organic solvent is n-hexane; Preferably, the polarity modifier comprises an alcoholic organic solvent; preferably, the polarity modifier comprises a C1-C4 monohydric alcohol and / or a C2-C4 polyhydric alcohol; more preferably, the polarity modifier comprises one or more of methanol, ethanol, ethylene glycol, 1,3-propanediol, and glycerol; even more preferably, the polarity modifier is methanol. More preferably, based on the total volume of the nonpolar organic solvent and the polar modifier being 100%, the volume percentage of the polar modifier is 0.1% to 10%, more preferably 0.5% to 5%.

6. The method according to any one of claims 1-5, characterized in that, The mixed phytosterols contain stigmasterol at a content of 10 wt% to 40 wt%, preferably 20 wt% to 30 wt%; and / or The total concentration of phytosterols in the feed solution is 1.0 g / L to 100.0 g / L, preferably 5.0 g / L to 50.0 g / L; and / or The mixed phytosterols include stigmasterol, β-sitosterol, campesterol, and brassosterol.

7. The method according to any one of claims 1-6, characterized in that, In the fractional extraction, the flow ratio of extractant to feed liquid is (0.5–5):1, preferably (0.8–2):1; and / or In the fractionation extraction, the flow ratio of detergent to feed liquid is (0.1–10):1, preferably (0.5–8):1, more preferably (7–8):1; and / or The detergent is of the same type as the raw material solvent in the raw material liquid.

8. The method according to any one of claims 1-7, characterized in that, The fractional extraction is a multi-stage fractional extraction, including an extraction section and a washing section; preferably, the extraction section of the multi-stage fractional extraction has 5 to 12 stages, more preferably 8 to 10 stages, and the washing section of the multi-stage fractional extraction has 5 to 20 stages, more preferably 10 to 15 stages; and / or The fractionation extraction is performed at a temperature of 10–70 °C, preferably 20–50 °C.

9. The method according to any one of claims 1-8, characterized in that, In step (2): The cooling crystallization includes: cooling the concentrated solution from 40–60 °C, preferably 45–50 °C, to -20–30 °C, preferably -20–10 °C, more preferably -10–0 °C; and / or The crystallization time is 10–60 min, preferably 20–30 min; and / or The back-extraction solvent used is a polar or non-polar organic solvent. The polar solvent includes one or more of alcohols, ketones, and esters. The non-polar organic solvent includes one or more of C6-C12 straight-chain alkanes, C6-C12 branched alkanes, C6-C12 cycloalkanes, and C1-C6 haloalkanes. Preferably, the back-extraction solvent includes one or more of the following: n-hexane, n-heptane, n-octane, n-nonane, n-decane, 2,2,4-trimethylpentane, isooctane, isodecanane, cyclohexane, methylcyclohexane, chloroform, and 1,2-dichloroethane. Preferably, the back-extraction solvent is n-hexane or 1,2-dichloroethane.

10. The method according to any one of claims 1-9, characterized in that, The method further includes the following steps: The mother liquor obtained after crystallization is concentrated under vacuum and then returned to the raw material liquid for reuse; or, the mother liquor obtained after crystallization is concentrated under vacuum and dissolved in the raw material solvent and then returned to the raw material liquid for reuse.