A method for separating ruscosapogenin epimers using ionic liquid liquid-liquid extraction

Abstract

The present application relates to the technical field of chemical separation, in particular to a method for separating the epimer of rusco-sapogenin by amino acid ionic liquid liquid-liquid extraction, wherein the amino acid ionic liquid is any one of 1-octyl-3-methyl imidazole-L-glutamate, 1-octyl-3-methyl imidazole-L-proline, 1-octyl-3-methyl imidazole-L-alanine, 1-octyl-3-methyl imidazole-L-histidine, 1-octyl-3-methyl imidazole-L-isoleucine, 1-octyl-3-methyl imidazole-L-lysine, 1-octyl-3-methyl imidazole-L-serine or a mixture of two or more thereof. The present application first uses specific amino acid ionic liquid to separate the epimer (R and S) of rusco-sapogenin, and the method has good separation effect, simple operation, high safety and can be applied to industrial large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical separation, in particular to a method for separating ruscosapogenin epimers by liquid-liquid extraction with amino acid ionic liquid. Background Art

[0002] Ruscogenin, whose molecular formula is C 27 H 42 O4, with a molecular mass of 430.63, is one of the main active ingredients of the commonly used Chinese medicinal herb Ophiopogon japonicus (Ophiopogon japonicus Ker-Gawl.) in clinical practice. Since 2010, the Pharmacopoeia of the People's Republic of China has used ruscosapogenin as a standard substance for the determination of total saponin content in Ophiopogon japonicus. It exhibits multiple biological activities, including cardiovascular protection, anti-inflammatory, anti-cell adhesion, hypoglycemic, anti-tumor, antioxidant, immune-enhancing, and liver and lung damage improvement, making it an excellent lead compound for the development of new steroidal saponin drugs. Currently, ruscosapogenin is only available from natural sources. However, naturally derived ruscosapogenin contains a pair of 25R and 25S isomers (their structures are shown below). The two have almost identical physical and chemical properties, differing only in optical rotation. Conventional methods are difficult to obtain optically pure 25R and 25S isomers, a problem that has hindered the pharmacological research and quality control of ruscosapogenin. Studies have shown that there are significant differences in the activities of 25R-ruscosapogenin and 25S-ruscosapogenin. Wu Yan et al. (Yu Boyang, Kou Junping, Wu Yan. Application of Ruscosapogenin Isomers in the Preparation of Drugs [P]. CN 107158007A.2017.) found that 25R-ruscosapogenin and 25S-ruscosapogenin have different pharmaceutical uses. 25R-ruscosapogenin can be used to treat or prevent acute lung injury. 25S-ruscosapogenin has inhibitory effects on various tumor cell lines such as MDA-MB-435, 95D, HepG2, Hela, MCF-7, and A549, while 25R-ruscosapogenin has no inhibitory effect on tumor cells. Therefore, it is of great significance to obtain optically pure ruscosapogenin through chiral separation technology. However, there are currently no reports on large-scale industrial separation methods for ruscosapogenin epimers, which hinders the development and utilization of ruscosapogenin as a new drug lead compound. Currently, the separation methods of epimers mainly include chemical separation, chromatographic separation, membrane separation, liquid-liquid extraction separation, etc. Among them, liquid-liquid extraction separation is the easiest to achieve industrialization. Chiral liquid-liquid extraction separation utilizes traditional liquid-liquid extraction technology to add a stereoselective chiral reagent to one or both phases of the liquid-liquid phase to achieve chiral recognition and separation of isomers. The chiral recognition effects include electrostatic effects, hydrogen bonding effects, van der Waals effects, etc. Compared with other separation technologies, liquid-liquid extraction has the advantages of easy industrialization, simple operation and high production capacity.

[0003]

[0004] Currently, only Chen Naidong et al. (Chen Naidong, Chen Qiong, He Jian. Preliminary study on the separation of 25R and 25S ruscosapogenin by high performance capillary electrophoresis [J]. Journal of Pharmaceutical Analysis, 2014(5):805-812.) reported using high performance capillary electrophoresis with hydroxypropyl-β-cyclodextrin as a chiral additive to separate a mixture of 25R and 25S ruscosapogenin diastereomers, achieving baseline separation with a resolution of 1.52±0.13. There are also reports on the use of supercritical fluid chromatography (Zhao, Y., McCauley, J., Pang, X., et al. Journal of Separation Science, 36(19), 3270–3276.) to separate structures similar to ruscosapogenin. However, these methods have low yields, narrow application ranges, high costs, and are difficult to achieve industrial production.

[0005] Ionic liquids are liquids composed entirely of ions that are molten at room temperature or under working conditions. Ionic liquids have unique properties such as being non-flammable, having good thermal stability, extremely low vapor pressure, being easily separated from other substances, and having good solubility in most organic or inorganic substances. They are good green solvents. Amino acid chiral ionic liquids are ionic liquids formed by introducing chiral centers through amino acids. They not only have the advantages of general ionic liquids, but also can produce chiral recognition effects based on the different binding forces with the two structures of the chiral compound. Compared with traditional resolving agents, the biggest advantage of amino acid chiral ionic liquids is that their structure can be designed as needed, the synthesis method is simple, and the ionic liquids can be adapted to different needs by combining different anions and cations. Researchers have applied amino acid ionic liquids to the resolution of chiral isomers. Fei et al. (Fei, T., Zhang, Q., Ren, D., et al. Journal of Chromatography A, 2010, 1217(28): 4669-4674.) used imidazole-based amino acid ionic liquids as both solvents and chiral selectors to study the resolution of four amino acid racemates. The results showed that amino acid ionic liquids had a good resolution effect on phenylalanine and tyrosine, with enantiomeric excesses (ee%) of 35.8% and 21.9%, respectively. Ll et al. (Ll, A., Ds, A., Fl, A., et al. Journal of Molecular Liquids, 2019, 294: 111599.) developed a two-phase system of choline amino acid ionic liquid / choline amino acid ionic liquid copper complex, which can be used as both phase-forming components and chiral selectors for chiral separation of valine enantiomers. The valine enantiomers were preferentially recognized in the phase rich in choline amino acid ionic liquid (upper phase), and the maximum ee value obtained in the upper phase was 55.6%.

[0006] As chiral resolving agents, ionic liquids exhibit structural selectivity. This means that to resolve a chiral compound, the ionic liquid first binds to both chiral structures and then achieves its resolution based on the differing binding strengths between the two structures. Therefore, selecting the appropriate ionic liquid is a technical challenge in this field. Not all ionic liquids possess chiral resolution properties; a suitable ionic liquid must possess different binding strengths for the two chiral molecules.

[0007] Currently, there is no report on the method of using ionic liquids as chiral selectors for the liquid-liquid extraction separation of ruscosapogenin diastereomers. Summary of the Invention

[0008] This invention, which falls within the scope of industrial separation, comprises a method for liquid-liquid extraction and separation of ruscosapogenin epimers using an amino acid ionic liquid as a chiral selector, which exhibits highly selective chiral recognition for ruscosapogenin epimers. This invention addresses the high cost and difficulty in industrializing existing liquid-liquid extraction and separation methods for ruscosapogenin epimers.

[0009] Provided is a method for resolving ruscosapogenin epimers by using an amino acid ionic liquid as a chiral selector, characterized in that the amino acid ionic liquid is 1-octyl-3-methylimidazole-L-glutamate, 1-octyl-3-methylimidazole-L-proline, 1-octyl-3-methylimidazole-L-alanine, 1-octyl-3-methylimidazole-L-arginine, 1-octyl-3-methylimidazole-L-histidine, 1-octyl-3-methylimidazole-L-isoleucine, 1-octyl-3-methylimidazole-L-lysine, or 1-octyl-3-methylimidazole-L-serine.

[0010] Furthermore, the method for liquid-liquid extraction and separation of ruscosapogenin epimers using the same as a chiral selector comprises the following steps:

[0011] 1) adding 40% by volume of methanol to water, and adding a certain concentration of 1-octyl-3-methylimidazolium-L-amino acid salt ionic liquid as an extractant, dissolving 50-200 ppm of a mixture of ruscosapogenin epimers in a water-immiscible organic solvent as a feed liquid, and extracting the feed liquid and the extractant by oscillation at room temperature for 12 hours, and allowing to stand for separation, wherein 25R-ruscosapogenin is enriched in the oil phase, and 25S-ruscosapogenin is enriched in the aqueous phase.

[0012] 2) After extraction and separation, the aqueous phase contains a small amount of 25S-ruscosapogenin, which can be recycled as an extractant. The oil phase is distilled to obtain 25R-ruscosapogenin.

[0013] 3) The aqueous phase needs to be regenerated after multiple cycles. After dichloromethane back extraction, the dichloromethane is distilled to obtain 25S-ruscosapogenin, and the amino acid ionic liquid in the water can be recycled.

[0014] Preferably, the water-immiscible organic solvent is 1,2-dichloroethane, butyl acetate, dichloromethane, or ethyl acetate.

[0015] Preferably, the concentration of the amino acid ionic liquid in the extractant is in the range of 0.01-0.16 mol / L.

[0016] Preferably, the concentration of the substance to be separated in the feed liquid is in the range of 50 ppm to 200 ppm.

[0017] Preferably, the types of amino acid ionic liquids in the extractant are 1-octyl-3-methylimidazole-L-glutamate, 1-octyl-3-methylimidazole-L-proline, 1-octyl-3-methylimidazole-L-alanine, 1-octyl-3-methylimidazole-L-arginine, 1-octyl-3-methylimidazole-L-histidine, 1-octyl-3-methylimidazole-L-isoleucine, 1-octyl-3-methylimidazole-L-lysine, and 1-octyl-3-methylimidazole-L-serine.

[0018] The substances to be separated are ruscosapogenin, natural derivatives and artificial derivatives of ruscosapogenin.

[0019] 1-Octyl-3-methylimidazolium-L-amino acid salt was synthesized via a displacement method. 1-Octyl-3-methylimidazolium bromide was first synthesized via quaternization, followed by the synthesis of the intermediate 1-Octyl-3-methylimidazolium base. Finally, the 1-Octyl-3-methylimidazolium-L-amino acid salt was synthesized via acid-base neutralization. After purification, the amino acid ionic liquid was characterized by infrared spectrophotometry and proton nuclear magnetic resonance spectroscopy.

[0020]

[0021] Synthesis of 1-octyl-3-methylimidazolium bromide

[0022] Measure 39.6 mL (0.5 mol, 1 eq) of N-methylimidazole, 108.5 mL (0.625 mol, 1.25 eq) of n-octane bromide, and an appropriate amount of isopropanol as the solvent. Heat at 70°C with stirring and reflux for 72 h. Remove the isopropanol by rotary evaporation at 55°C to obtain a yellow, viscous liquid. Add an equal volume of water and extract five times with an equal volume of ethyl acetate. Collect the aqueous layer, remove the solvent by rotary evaporation, and dry under vacuum to obtain [OMIM]Br.

[0023] Synthesis of 1-octyl-3-methylimidazole base

[0024] [OMIM]Br (1.0 eq) and KOH (1.0 eq) were dissolved in an appropriate amount of anhydrous ethanol. The [OMIM]Br anhydrous ethanol solution was slowly added dropwise to the KOH anhydrous ethanol solution and stirred evenly. During the addition, a white turbid precipitate was rapidly generated in the reaction system. The reaction was carried out under ice-water bath conditions for 6 h. The 1-octyl-3-methylimidazole base anhydrous ethanol solution was filtered and used directly in the next reaction.

[0025] Synthesis of 1-octyl-3-methylimidazole-L-amino acid salt

[0026] L-amino acid (1.0 eq) was dissolved in an appropriate amount of anhydrous ethanol, and the L-amino acid anhydrous ethanol solution was slowly added dropwise to the anhydrous ethanol solution of 1-octyl-3-methylimidazole base (1.0 eq) and stirred evenly. The mixture was reacted in an ice-water bath for 12 hours. After removing the anhydrous ethanol by rotary evaporation, an equal volume of methanol-acetonitrile (9:1, v / v) was added and stirred evenly. The unreacted amino acid was precipitated, filtered, and the solvent was removed by rotary evaporation. The mixture was vacuum dried to obtain a viscous amino acid chiral ionic liquid.

[0027] 1 H-NMR Characterization of Amino Acid Ionic Liquids

[0028] 1H NMR (300 MHz, DMSO-d6) spectral data of the ionic liquid [Omim][L-Ala]: δ 9.71 (s, 1H), 7.86 (s, 1H), 7.79 (s, 1H), 4.19 (t, J = 7.13 Hz, 2H), 3.88 (s, 3H), 2.95 (q, J = 6.73 Hz, 1H), 1.77 (m, 2H), 1.22 (s, 10H), 1.05 (m, 3H), 0.83 (t, J = 6.76 Hz, 3H).

[0029]

[0030] 1-Octyl-3-methylimidazolium-L-alanine salt

[0031] [OMIM][L-Ala]

[0032] Ionic liquid [Omim][L-Arg] 1 H NMR (300 MHz, DMSO-d6) spectral data: δ 7.57 (s, 1H), 7.10 (s, 1H), 6.86 (s, 1H), 4.17 (t, J = 7.17 Hz, 2H), 3.93 (m, 1H), 3.85 (m, 2H), 3.85 (s, 3H), 3.05 (m, 3H), 1.76 (m, 2H), 1.40 (m, 2H), 1.24 (s, 12H), 0.85 (t, J = 6.94 Hz, 3H).

[0033]

[0034] 1-Octyl-3-methylimidazole-L-arginine salt

[0035] [OMIM][L-Arg]

[0036] Ionic liquid [Omim][L-Glu] 1H NMR (300 MHz, DMSO-d6) spectral data: δ 9.68 (s, 1H), 7.82 (s, 1H), 7.77 (s, 1H), 4.17 (t, J = 7.19 Hz, 2H), 3.88 (s, 3H), 2.50 (m, 1H), 1.95 (t, J = 7.17 Hz, 2H), 1.77 (m, 2H), 1.23 (s, 10H), 0.85 (t, J = 6.70 Hz, 3H).

[0037]

[0038] 1 -octyl-3 -methylimidazolium L-glutamate

[0039] [OMIM][L-Glu]

[0040] [Omim][L-His] of the ionic liquid 1 H NMR (300 MHz, DMSO-d6) spectral data: δ 9.62 (s, 1H), 7.84 (s, 1H), 7.77 (s, 1H), 7.45 (s, 1H), 6.68 (s, 1H), 4.16 (t, J = 7.20 Hz, 2H), 3.86 (s, 3H), 3.18 (t, J = 4.04 Hz, 1H), 2.95 (dd, J = 14.30, 3.73 Hz, 1H), 2.48 (dd, J = 14.30, 8.72 Hz, 1H), 1.75 (m, 2H), 1.21 (s, 10H), 0.82 (t, J = 6.69, 3H).

[0041]

[0042] 1 -octyl-3 -methylimidazolium L-histidine

[0043] [OMIM][L-His]

[0044] [Omim][L-Ile] of the ionic liquid 1 H NMR (300 MHz, DMSO-d6) spectral data: δ 9.72 (s, 1H), 7.85 (s, 1H), 7.78 (s, 1H), 4.19 (t, J = 7.22 Hz, 2H), 3.88 (s, 2H), 2.77 (d, J = 4.29 Hz, 1H),

[0045] 1.77 (m, 2H), 1.58 (m, 1H), 1.37 (m, 1H), 1.22 (s, 10H), 0.79 (m, 6H).

[0046]

[0047] 1 -octyl-3-methylimidazolium-L-isoleucinate

[0048] [OMIM][L-Ile]

[0049] [Omim][L-Lys] of ionic liquid 1 H NMR (300 MHz, DMSO-d6) spectral data: δ 9.68 (s, 1H), 7.84 (s, 1H), 7.77 (s, 1H), 4.18 (t, J = 7.21 Hz, 2H), 3.87 (s, 3H), 2.80 (m, 1H), 2.47 (m, 2H), 1.80 (m, 2H), 1.23 (s, 16H), 0.84 (t, J = 6.94 Hz, 3H).

[0050]

[0051] 1 -octyl-3-methylimidazolium-L-lysinate

[0052] [OMIM][L-Lys]

[0053] [Omim][L-Pro] of ionic liquid 1 H NMR (300 MHz, DMSO-d6) spectral data: δ 9.02 (s, 1H), 7.63 (t, J = 1.64 Hz, 1H), 7.57 (t, J = 1.64 Hz, 1H), 4.10 (t, J = 7.23, 2H), 3.81 (s, 3H), 3.27 (dd, J = 8.10, 6.08 Hz, 1H), 2.94 (dt, J = 10.61, 6.75 Hz, 1H), 2.76 (dt, J = 10.61, 6.75 Hz, 1H), 1.90 (m, 1H), 1.73 (m, 2H), 1.55 (m, 4H), 1.17 (s, 10H), 0.79 (t, J = 6.68 Hz, 3H).

[0054]

[0055] 1 -octyl-3-methylimidazolium-L-proline

[0056] [OMIM][L-Pro]

[0057] [Omim][L-Ser] of ionic liquid 1H NMR (300 MHz, DMSO-d6) spectral data: δ9.51 (s, 1H), 7.83 (t, J = 1.66 Hz, 1H), 7.76 (t, J = 1.66 Hz, 1H), 4.18 (t, J = 4.18 Hz, 2H), 3.87 (s, 3H), 3.64 (s, 1H), 3.28 (d, J = 7.20 Hz, 2H), 2.89 (t, J = 7.16 Hz, 1H), 1.77 (m, 2H), 1.24 (s, 10H), 0.84 (t, J = 6.71 Hz, 3H).

[0058]

[0059] 1-Octyl-3-methylimidazole-L-serine salt

[0060] [OMIM][L-Ser]

[0061] Infrared detection results:

[0062] Dry potassium bromide tablets, add amino acid ionic liquid and intermediates dropwise into KBr slices and measure their wavenumbers in the range of 400-4000 cm -1 Infrared absorption in the range, such as Figure 2 As shown in Table 1. The infrared absorption of the intermediate [OMIM]Br is as follows: 3096 cm -1 The CH stretching vibrations on the imidazole group are 2956, 2927, and 2857 cm -1 The CH stretching vibration of the saturated fatty chain, 1635, 1466 cm -1 The C=C stretching vibration of the imidazole ring skeleton is 1573 cm -1 The C=N stretching vibration of the imidazole skeleton is 1167 cm -1 is the in-plane deformation vibration of the imidazole ring CH, 3150cm -1 The weak absorption at [OMIM]Br is due to C-Br stretching vibrations, confirming the successful synthesis of [OMIM]Br. The infrared spectral data for the amino acid ionic liquid are shown in Table 1. The C=O stretching vibrations all shift toward lower frequencies, indicating a strong attraction at this functional group, confirming the successful synthesis of the amino acid ionic liquid.

[0063] Table 1 Infrared characteristic peaks of various amino acid ionic liquids

[0064]

[0065] The present invention synthesizes a series of amino acid ionic liquids, which are used for the separation of ruscosapogenin epimers for the first time. Figure 1 a shows that different organic solvents have different separation effects, and dichloromethane is the best solvent; Figure 1b shows that the concentration of amino acid ionic liquid also has an effect on the separation effect, and it is nonlinear. Among the amino acid ionic liquid concentrations of 0.01mol / L, 0.02mol / L, 0.04mol / L, 0.08mol / L, and 0.16mol / L, the effect is best when it is 0.04mol / L. The concentration of ruscosapogenin diastereomer also has an effect on the selectivity coefficient. Figure 1 c shows that the effect is best when the concentration of ruscosapogenin diastereomer is 100 ppm, and the relationship between concentration and effect is nonlinear; Figure 1 Figure d shows that different amino acid ionic liquids have different separation degrees for ruscosapogenin. 1-octyl-3-methylimidazolium-L-glutamate is the most effective chiral selector, followed by 1-octyl-3-methylimidazolium-L-proline. 1-octyl-3-methylimidazolium-L-arginine has a selectivity coefficient below 1, indicating a lack of separation. Other separation effects are average, with selectivity coefficients above 1 but below 1.5. This further demonstrates that ionic liquids, as chiral resolving agents, are structurally selective, and not all ionic liquids can achieve the objectives of the present invention.

[0066] Beneficial effects

[0067] Compared with the prior art, the innovation of the present invention is:

[0068] The present invention uses a specific amino acid ionic liquid to separate ruscosapogenin diastereomers (R and S) for the first time. The method has good separation effect, simple operation, high safety, and can be applied to industrial large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 The influence of different factors on the separation of ruscosapogenin epimers;

[0070] a: Effect of hydrophobic organic solvent type on the partition coefficient and selectivity coefficient; b: Effect of amino acid ionic liquid concentration on the selectivity coefficient;

[0071] c: Effect of ruscosapogenin diastereomer concentration on selectivity coefficient; d: Effect of amino acid ionic liquid type on selectivity coefficient; Figure 2 IR spectra of amino acid ionic liquids. DETAILED DESCRIPTION

[0072] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. The scope of protection of the present invention is not limited by the embodiments, but is determined by the claims. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0073] During the operation of the present invention, 40% by volume of methanol is added to water, and a certain concentration of 1-octyl-3-methylimidazole-L-amino acid salt ionic liquid is added as an extraction agent. A 50-200 ppm mixture of ruscosapogenin epimers is dissolved in a water-immiscible organic solvent as a feed liquid. The feed liquid and the extraction agent are mixed in a centrifuge tube, and the mixture is shaken and extracted on a shaking table at room temperature for 12 hours, followed by standing and separation. The oil phase is taken and analyzed for the concentration of the ruscosapogenin epimers. The concentration of the ruscosapogenin epimers is analyzed and detected by high performance liquid chromatography on a reverse phase chromatography column. The concentration of the ruscosapogenin epimers in the aqueous phase can be obtained by subtracting the total concentration of the single isomers and the concentration in the oil phase according to the law of conservation of mass. The distribution coefficient and selectivity coefficient of 25R-ruscosapogenin and 25S-ruscosapogenin can be obtained by the following formulas:

[0074]

[0075]

[0076]

[0077] where K 25R and K 25S are the partition coefficients of 25R-ruscosaponin and 25S-ruscosaponin, c 25Ro and c 25Rw are the concentrations of 25R-ruscosapogenin in the oil phase and the water phase, c 25So and c 25Sw where α is the selectivity coefficient, and α is the concentration of 25S-ruscosapogenin in the oil phase and the aqueous phase, respectively. The selectivity coefficient must be greater than 1. A larger selectivity coefficient indicates a better separation effect of 25R-ruscosapogenin and 25S-ruscosapogenin.

[0078] The pretreatment method of the high performance liquid chromatography analysis sample in the embodiment is to accurately transfer 1 mL of the oil phase and evaporate it, reconstitute it with 1 mL of methanol and inject it into the activated C18-SPE, elute it with 6 column volumes of 60% volume percentage of methanol aqueous solution, discard this part, elute it with methanol for 6 column volumes, collect the methanol elution part, evaporate it, make up to 1 mL with methanol and pass it through a 0.22 μm microporous membrane for high performance liquid chromatography analysis.

[0079] In the embodiment, high performance liquid chromatography was used to quantitatively analyze the concentration of ruscosapogenin diastereomers. The specific analysis conditions of the high performance liquid chromatography were as follows: acetonitrile-water (added with 8 mmol / L 1-octyl-3-methylimidazolium-L-glutamate ionic liquid and 4 mmol / L copper sulfate pentahydrate) as the mobile phase, a chromatographic column was a Hiber Purospher STAR RP-18e (250 mm × 4.6 mm, 5 μm), an injection volume of 10 μL, a flow rate of 0.4 mL / min, a column temperature of 20° C., an evaporative light scattering detector, a drift tube temperature of 100° C., and a nebulizing gas flow rate of 2.0 L / min.

[0080] Example 1:

[0081] The effect of the type of hydrophobic organic solvent on the distribution coefficient and selectivity coefficient was investigated.

[0082] Prepare a 40% volume percent methanol-water solution. Weigh a certain amount of 1-octyl-3-methylimidazolium-L-proline salt and dilute it to an ionic liquid concentration of 0.04 mol / L as the extractant. Weigh a certain amount of a mixture of ruscosapogenin epimers and dilute it to a total concentration of 100 ppm with 1,2-dichloroethane, butyl acetate, dichloromethane, and ethyl acetate to form the feed solution. Combine 10 mL of the feed solution and 10 mL of the extractant in a centrifuge tube and shake on a shaker at room temperature for 12 hours. After extraction, allow the mixture to stand and separate. Once the interface between the two phases is clear, precisely aspirate 1 mL of the oil phase for pre-treatment and high-performance liquid chromatography analysis. The concentrations of 25R-ruscosapogenin and 25S-ruscosapogenin are determined, and the distribution coefficient and selectivity are calculated.

[0083] See attached for the results Figure 1 a. Dichloromethane is the best solvent, with the distribution coefficients of 25R-ruscosapogenin and 25S-ruscosapogenin being 7.19 and 4.36, respectively, and the selectivity coefficient being 1.65. The separation effects of 1,2-dichloroethane and ethyl acetate are intermediate; the separation effect of butyl acetate is the worst.

[0084] Example 2: The effect of the concentration of amino acid ionic liquid on the partition coefficient and selectivity coefficient was investigated.

[0085] Prepare 40% volume percentage methanol aqueous solution, take a certain amount of 1-octyl-3-methyl imidazole-L-proline salt, and use the above solution to make up to an ionic liquid concentration of 0.01 mol / L, 0.02 mol / L, 0.04 mol / L, 0.08 mol / L, 0.16 mol / L as an extractant. Take a certain amount of rusco sapogenin epimer mixture, and use dichloromethane to make up to a total rusco sapogenin epimer mixture concentration of 100 ppm as a feed liquid. Take 10 mL of the feed liquid and 10 mL of the extractant, mix them in a centrifuge tube, and shake at room temperature for 12 hours. After the extraction is completed, separate and stand until the two-phase interface is clear. Precisely take 1 mL of the oil phase, and perform high performance liquid chromatography analysis after pretreatment to detect the concentrations of 25R-rusco sapogenin and 25S-rusco sapogenin, and calculate the distribution coefficient and the selectivity coefficient.

[0086] The results are shown in the following table: Figure 1 b, the effect is best when the amino acid ionic liquid concentration is 0.04 mol / L, the distribution coefficients of 25R-rusco sapogenin and 25S-rusco sapogenin are 6.62 and 4.42 respectively, and the selectivity coefficient is 1.50.

[0087] Example 3: The effect of the concentration of rusco sapogenin epimers on the distribution coefficient and the selectivity coefficient was investigated.

[0088] Prepare 40% volume percentage methanol aqueous solution, take a certain amount of 1-octyl-3-methyl imidazole-L-proline salt, and use the above solution to make up to an ionic liquid concentration of 0.04 mol / L as an extractant. Take a certain amount of rusco sapogenin epimer mixture, and use dichloromethane to make up to a total rusco sapogenin epimer mixture concentration of 50 ppm, 100 ppm, 200 ppm as a feed liquid. Take 10 mL of the feed liquid and 10 mL of the extractant, mix them in a centrifuge tube, and shake at room temperature for 12 hours. After the extraction is completed, separate and stand until the two-phase interface is clear. Precisely take 1 mL of the oil phase, and perform high performance liquid chromatography analysis after pretreatment to detect the concentrations of 25R-rusco sapogenin and 25S-rusco sapogenin, and calculate the distribution coefficient and the selectivity coefficient.

[0089] The results are shown in the following table: Figure 1 c, the effect is best when the rusco sapogenin epimer concentration is 100 ppm, the distribution coefficients of 25R-rusco sapogenin and 25S-rusco sapogenin are 6.60 and 3.28 respectively, and the selectivity coefficient is 2.01.

[0090] Example 4:

[0091] The effect of the type of amino acid ionic liquid on the distribution coefficient and the selectivity coefficient was investigated.

[0092] A 40% volume percentage methanol aqueous solution was prepared, and a certain amount of 1-octyl-3-methylimidazolium-L-glutamate, 1-octyl-3-methylimidazolium-L-proline, 1-octyl-3-methylimidazolium-L-alanine, 1-octyl-3-methylimidazolium-L-arginine, 1-octyl-3-methylimidazolium-L-histidine, 1-octyl-3-methylimidazolium-L-isoleucine, 1-octyl-3-methylimidazolium-L-lysine, and 1-octyl-3-methylimidazolium-L-serine were weighed, and the above solution was used to make up the solution to a concentration of 0.04 mol / L as an extractant. A certain amount of a mixture of rusco sapogenin epimers was weighed and made up with dichloromethane to a total concentration of 100 ppm as a feed solution. 10 mL of the feed solution and 10 mL of the extractant were mixed in a centrifuge tube, and shaken at room temperature for 12 hours. After extraction was completed, the two phases were separated, and when the interface was clear, 1 mL of the oil phase was precisely taken and subjected to pretreatment for high performance liquid chromatography analysis to detect the concentrations of 25R-rusco sapogenin and 25S-rusco sapogenin, and the distribution coefficient and the selectivity coefficient were calculated.

[0093] The results are shown in the following table Figure 1 d, 1-octyl-3-methylimidazolium-L-glutamate had the best effect as a chiral selector, followed by 1-octyl-3-methylimidazolium-L-proline. The selectivity coefficient of 1-octyl-3-methylimidazolium-L-arginine was less than 1, indicating that it did not have a separation effect. The other separation effects were general, and the selectivity coefficient was higher than 1 but less than 1.5.

[0094] When 1-octyl-3-methylimidazolium-L-glutamate was used as an extractant, the distribution coefficients of 25R-rusco sapogenin and 25S-rusco sapogenin were 6.30 and 2.79, respectively, and the selectivity coefficient was 2.26.

[0095] Example 5: According to the optimal conditions selected above, a multi-stage cross-flow extraction method was used to prepare pure rusco sapogenin epimers.

[0096] Formulate 40% volume percentage of methanol aqueous solution, take a certain amount of 1-octyl-3-methyl imidazole-L glutamic acid salt with the above solution to constant volume to the ionic liquid concentration of 0.04 mol / L as the extractant. Take a certain amount of rusco sapogenin epimer mixture with dichloromethane to constant volume to the total concentration of rusco sapogenin epimer mixture of 200 ppm as the feed liquid. Take the feed liquid, extractant, etc. Mix in the separatory funnel at room temperature for 12 hours. After the extraction is completed, separate and wait for the two-phase interface to be clear, and then accurately take 1 mL of the oil phase for pretreatment and high performance liquid chromatography analysis to detect the concentration of 25R-rusco sapogenin and the concentration of 25S-rusco sapogenin, and calculate the purity of 25R-rusco sapogenin in the oil phase. Separate the aqueous phase and mix with an equal volume of fresh extractant in the separatory funnel, and repeat the above steps 10 times. The purity of 25R-rusco sapogenin in the oil phase after each stage of extraction is 74.73%, 74.94%, 77.08%, 78.66%, 81.09%, 81.89%, 83.75%, 84.78%, 86.64%, and 87.35%, respectively. It shows that the method has good separation effect, is feasible and stable.

Claims

1. A method for separating ruscosapogenin epimers by liquid-liquid extraction using amino acid ionic liquid as a chiral selector, characterized in that: The amino acid ionic liquid is any one of 1-octyl-3-methylimidazole-L-glutamate, 1-octyl-3-methylimidazole-L-alanine, 1-octyl-3-methylimidazole-L-histidine, 1-octyl-3-methylimidazole-L-isoleucine, 1-octyl-3-methylimidazole-L-lysine, and 1-octyl-3-methylimidazole-L-serine, or a mixture of any two or more thereof.

2. The method according to claim 1, characterized in that The water-immiscible organic solvent in the liquid-liquid extraction is any one of 1,2-dichloroethane, butyl acetate, dichloromethane, and ethyl acetate, or a mixture of any two or more thereof.

3. The method according to claim 1, wherein The concentration range of amino acid ionic liquid in liquid-liquid extraction was 0.01-0.16 mol / L.

4. The method according to claim 1, wherein The concentration range of the substance to be separated is 50 ppm-200 ppm; the substance to be separated is ruscosapogenin.

5. The method according to claim 4, characterized in that The ruscosapogenin is a natural derivative or an artificial derivative of ruscosapogenin.

6. The method according to any one of claims 1 to 5, characterized in that Follow these steps to achieve this: 1) Methanol is added to water, and an amino acid salt ionic liquid is added as an extractant. A 50-200 ppm mixture of ruscosapogenin diastereomers is dissolved in a water-immiscible organic solvent as a feed solution. The feed solution and the extractant are shaken and extracted at room temperature for 12 hours. The mixture is allowed to stand for separation, and 25R-ruscosapogenin is enriched in the oil phase, while 25S-ruscosapogenin is enriched in the aqueous phase. 2) After extraction and separation, the aqueous phase is supplemented with methanol and step 1 is repeated). The oil phase is distilled to obtain 25R-ruscosapogenin; 3) The aqueous phase is back-extracted with dichloromethane and the dichloromethane is distilled to obtain 25S-ruscosapogenin.

7. The method according to claim 6, wherein A 40% volume percent methanol aqueous solution was prepared, 1-octyl-3-methylimidazolium-L-glutamate was weighed and diluted with the above solution to an ionic liquid concentration of 0.04 mol / L as an extractant; a mixture of ruscosapogenin epimers was weighed and diluted with dichloromethane to a total concentration of the mixture of ruscosapogenin epimers of 200 ppm as a feed liquid; the feed liquid and the extractant were mixed in equal proportions in a separatory funnel and shaken on a shaker at room temperature for 12 hours; after the extraction was completed, the oil phase and the aqueous phase were allowed to stand and separate, wherein 25R-ruscosapogenin was enriched in the oil phase and 25S-ruscosapogenin was enriched in the aqueous phase, and the solvent was recovered to obtain 25R-ruscosapogenin and 25S-ruscosapogenin.

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