Separation and purification method of lanosterol crude product

Abstract

The invention discloses a separation and purification method of a lanosterol crude product. The method comprises the following steps: separating and purifying a lanosterol crude product solution by using preparative liquid chromatography to obtain a lanosterol pure product; wherein a mobile phase used for preparing the liquid chromatogram comprises a solvent 1 and acetonitrile; the solvent 1 is selected from one or more of methanol, ethanol, normal propyl alcohol, isopropanol and dichloromethane. The method disclosed by the invention has relatively good selectivity on separation and purification of lanosterol, the process is simple, the implementation is easy, the separation period is short, the separated lanosterol is high in purity and high in yield, efficient separation of lanosterol and dihydrolanosterol is realized, and the method has important significance on preparation of lanosterol monomers.

Description

[0001] This application claims priority to Chinese patent application No. CN2023106723234 filed on June 7, 2023. The full text of the above-mentioned Chinese patent application is hereby cited as part of this application. Technical Field

[0002] The invention relates to the technical field of organic matter purification, and in particular to a method for separating and purifying crude lanosterol. Background Art

[0003] Lanosterol is also known as isocholesterol. Its chemical name is (3β)-8,24-lanostadien-3-ol. It is a white crystal belonging to the tetracyclic triterpenoids. It has many important biological activities and a wide range of pharmacological effects, such as anti-cancer, lowering blood pressure, treating leukemia, lowering blood sugar, and alleviating cataracts in experimental animals (rabbits and dogs). Lanosterol is an intermediate in the biosynthesis of sterols and can also be used as a raw material for cosmetics. It is an important precursor of ergosterol and a variety of triterpenoids with biological activities such as anti-inflammatory, anti-cancer, and hypolipidemic. Therefore, it is of high research value to separate and purify lanosterol with high purity from crude lanosterol. In the crude lanosterol available on the market, the ratio of lanosterol to dihydrolanosterol is about 2:1. Because the molecular structures of the two are very similar and their polarities are very close, it is difficult to separate them using conventional methods.

[0004] At present, there are bromination, extraction, molecular distillation and preparative chromatography methods for the extraction and separation of lanosterol. The reagents used in the classic bromination method are expensive and the recovery rate is low. The traditional extraction method (Zhang Xinghua, Separation and Extraction Process of Cholesterol in Lanolin, Master's Thesis of Tianjin University, 2010) obtains crude lanosterol and dihydrolanosterol from lanolin by solvent crystallization, without further separation and purification, and the product purity is poor; the molecular distillation method (CN101817859A, Separation and Extraction Method of Cholesterol in Lanolin Alcohol) obtains 63-70% of byproduct lanosterol by molecular distillation and then selectively crystallizes the solvent, and the product purity is poor; the preparative chromatography method (CN101691391A, Method for Separation and Purification of Crude Lanosterol) can quickly obtain high-purity lanosterol, but the processing volume is small and the equipment cost is high. It is limited to laboratory research and difficult to achieve industrial production. Therefore, the research on this technology still has a large room for development. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for separating and purifying a crude lanosterol product. The method has good selectivity, a simple separation operation process, is easy to implement, has a short separation period, a high yield, and the obtained lanosterol product has high purity, realizes efficient separation of lanosterol and dihydrolanosterol, can carry out large-scale separation and purification of the crude lansterol product, meets the demand for large-scale preparation of lansterol, and is of great significance to the preparation of lansterol monomer.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for separating and purifying crude lanosterol comprises: separating and purifying a crude lanosterol solution by preparative liquid chromatography to obtain pure lanosterol;

[0008] Wherein, the mobile phase used in the preparative liquid chromatography comprises solvent 1 and acetonitrile;

[0009] Wherein, the solvent 1 is selected from one or more of methanol, ethanol, n-propanol, isopropanol and dichloromethane.

[0010] The crude lanosterol of the present invention contains lanosterol and dihydrolanosterol.

[0011] In some embodiments, in the crude lanosterol, the mass ratio of lanosterol to dihydrolanosterol is (1.5-2.5):1.

[0012] According to some embodiments of the present invention, in the mobile phase, the volume ratio of solvent 1 to acetonitrile is 1:(1-10), for example 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10.

[0013] According to some embodiments of the present invention, the flow rate of the mobile phase in the liquid chromatography column is 1 to 6 L / min, for example, 1 L / min, 2 L / min, 3 L / min, 4 L / min, 5 L / min or 6 L / min.

[0014] According to some embodiments of the present invention, the solvent of the crude lanosterol solution is an organic solvent, preferably, the organic solvent is any one or a combination of methanol, ethanol, n-propanol, isopropanol, dichloromethane, acetone, N,N-dimethylformamide. In some embodiments, the mass volume ratio of the crude lanosterol to the organic solvent is 12:1 to 15:1 (g / L), for example, 12.5:1, 13:1, 13.5:1, 14:1 or 14.5:1.

[0015] According to some embodiments of the present invention, the preparative liquid chromatography is a preparative liquid chromatography in a dynamic axial compression column mode. In some embodiments, the packing of the dynamic axial compression column is octadecyl reverse phase silica gel. Preferably, the silica gel particle size is 8 to 12 μm. Preferably, the inner diameter of the dynamic axial compression column is 400 to 1200 mm. Preferably, the packing height is 200 to 300 mm.

[0016] According to some embodiments of the present invention, the detection wavelengths of the preparative liquid chromatography ultraviolet detector are 210 nm and 245 nm.

[0017] According to some embodiments of the present invention, the method further comprises: collecting target component fractions by using the preparative liquid chromatography, and performing the steps of removing solvent, recrystallizing and drying the collected fractions.

[0018] In some embodiments of the present invention, the pressure during the removal of the solvent is -0.01 to -0.08 MPa.

[0019] In some embodiments of the present invention, the temperature during the removal of the solvent is 40-45°C.

[0020] In some embodiments of the present invention, the solvent used for recrystallization is an alcohol solvent, such as methanol, ethanol, n-propanol, isopropanol, preferably methanol. Preferably, the volume mass ratio of the alcohol solvent to the crude lanosterol is (5-9):1 (L / kg), such as 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, 8:1, 8.5:1 or 8.8:1.

[0021] In some embodiments of the present invention, the drying pressure is -0.01 to -0.08 MPa.

[0022] In some embodiments of the present invention, the drying temperature is 40-45°C.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The invention uses preparative liquid chromatography to separate and purify crude lanosterol, and realizes efficient separation of lanosterol and dihydrolanosterol by optimizing process parameters such as mobile phase composition and flow rate. The method of the invention has good selectivity, simple separation operation process, easy implementation and short separation cycle, the purity of lanosterol can be as high as more than 99%, and the yield reaches more than 92%, which meets the research and development needs in the medical field and the needs of mass preparation of lanosterol. DETAILED DESCRIPTION

[0025] In order to make the purpose and technical solution of the present invention more clear, the present invention is further described in detail below in conjunction with the embodiments. The specific embodiments described here are only used to explain the present invention and are not intended to constitute any limitation to the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0027] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0028] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0029] The term "pure" as used herein means a purity of ≥ 90%. In some embodiments, the term "pure" means a purity of ≥ 95%. In other embodiments, the term "pure" means a purity of ≥ 98%.

[0030] The solvents used in the following examples of the present invention can be obtained from commercial sources.

[0031] The crude lanosterol used in the following examples of the present invention is a commercially available product, containing about 60 wt % lanosterol and about 35 wt % dihydrolanosterol, and the supplier is Shandong Junrui Co., Ltd. or Sibaiquan Chemical (Shanghai) Co., Ltd.

[0032] The model of the preparative liquid chromatograph in the dynamic axial compression column mode used in the following examples of the present invention is:

[0033] DAC 600.

[0034] Example 1

[0035] This embodiment provides a method for separating and purifying crude lanosterol, comprising the following steps:

[0036] (1) Dissolve 450 g of crude lanosterol in 30 L of methanol and stir to dissolve to prepare a crude lanosterol solution.

[0037] (2) 50 kg of C18 filler with a particle size of 10 μm was added with 140 L of homogenization reagent methanol, and the DAC column system pressure was set. After stirring at room temperature for 1.5 hours, the homogenate was sent to a dynamic axial compression column with a diameter of 400 mm. After the mobile phase was removed, the column filler was compacted to complete the filling of the DAC column; the dynamic axial compression column was continuously flushed with the mobile phase for 15-20 minutes until the chromatograph baseline was stable; the wavelength of the UV detector was set to 210 nm and 245 nm, and the flow rate of the infusion pump was set to 1 L / min. The sample solution was filtered with a 0.45 μm filter and then injected into the balanced dynamic axial compression column with an injection volume of 24 L. The sample separation was detected by a UV detector. The mobile phase was methanol:acetonitrile (V / V) = 1:1, and the elution time was 70 min.

[0038] (3) Using the preparative liquid chromatography system in step (2) to treat the crude lanosterol solution prepared in step (1), collecting the target component fractions according to the chromatogram, removing the solvent from the collected fractions at -0.08 MPa and 40° C., then adding 2.5 L of methanol to dissolve, stirring and filtering, and drying the obtained solid matter at -0.08 MPa and 40° C. to obtain 250 g of pure lanosterol.

[0039] The purity of lanosterol was detected by high performance liquid chromatography. After detection, the purity of lanosterol reached 99.20% and the yield reached 92.59%.

[0040] Example 2

[0041] This embodiment provides a method for separating and purifying crude lanosterol, comprising the following steps:

[0042] (1) Dissolve 360 ​​g of crude lanosterol in 30 L of isopropanol and stir to dissolve to prepare a crude lanosterol solution.

[0043] (2) 50 kg of C18 filler with a particle size of 8 μm was added with 140 L of isopropanol as a homogenizing agent, and the DAC column system pressure was set. After stirring at room temperature for 1.5 hours, the homogenate was sent to a dynamic axial compression column with a diameter of 400 mm. After the mobile phase was removed, the column filler was compacted to complete the filling of the DAC column; the dynamic axial compression column was continuously flushed with the mobile phase for 15-20 minutes until the chromatograph baseline was stable; the wavelength of the UV detector was set to 210 nm and 245 nm, and the flow rate of the infusion pump was set to 1 L / min. After filtering the sample solution with a 0.45 μm filter, it was injected into the balanced dynamic axial compression column with an injection volume of 24 L. The sample separation was detected by a UV detector. The mobile phase was isopropanol:acetonitrile (V / V) = 1:10, and the elution time was 70 min.

[0044] (3) The crude lanosterol solution prepared in step (1) was treated with the preparative liquid chromatography system in step (2), and the target component fractions were collected according to the chromatogram. The collected fractions were desolvated at -0.08 MPa and 40° C., and then 2 L of methanol was added to dissolve, stirred and filtered. The obtained solid matter was dried at -0.08 MPa and 40° C. to obtain 199 g of pure lanosterol.

[0045] The purity of lanosterol was detected by high performance liquid chromatography. After detection, the purity of lanosterol reached 99.01% and the yield reached 92.13%.

[0046] Comparative Example 1

[0047] This comparative example provides a method for separating and purifying crude lanosterol, which is different from Example 1 in that the mobile phase in the chromatographic conditions in step (2) is methanol and ethyl acetate.

[0048] The results showed that adjusting the mobile phase ratio and flow rate could not effectively separate lanosterol and dihydrolanosterol.

[0049] Comparative Example 2

[0050] This comparative example provides a method for separating and purifying crude lanosterol, which is different from Example 1 in that the mobile phase in the chromatographic conditions in step (2) is dichloromethane and ethyl acetate.

[0051] The results showed that adjusting the mobile phase ratio and flow rate could not effectively separate lanosterol and dihydrolanosterol.

[0052] Comparative Example 3

[0053] This comparative example provides a method for separating and purifying crude lanosterol, which is different from Example 1 in that the mobile phase in the chromatographic conditions in step (2) is methanol and dichloromethane.

[0054] The results showed that adjusting the mobile phase ratio and flow rate could not effectively separate lanosterol and dihydrolanosterol.

[0055] Although the present invention has been described in detail above by means of general description, specific implementation methods and tests, it is obvious to those skilled in the art that some modifications or improvements may be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for separating and purifying crude lanosterol, comprising: Using preparative liquid chromatography to separate and purify the crude lanosterol solution to obtain pure lanosterol; Wherein, the mobile phase used in the preparative liquid chromatography comprises solvent 1 and acetonitrile; The solvent 1 is selected from one or more of methanol, ethanol, n-propanol, isopropanol and dichloromethane.

2. The method according to claim 1, characterized in that In the mobile phase, the volume ratio of solvent 1 to acetonitrile is 1:(1-10).

3. The method according to claim 1 or 2, characterized in that: The flow rate of the mobile phase in the liquid chromatography column is 1 to 6 L / min.

4. The method according to any one of claims 1 to 3, characterized in that: The crude lanosterol includes lanosterol and dihydrolanosterol.

5. The method according to any one of claims 1 to 4, characterized in that: The solvent of the crude lanosterol solution is selected from one or more of methanol, ethanol, n-propanol, isopropanol, dichloromethane, acetone, and N,N-dimethylformamide. Preferably, the mass volume ratio of the crude lanosterol to the solvent is (12g-15g):1L.

6. The method according to claim 5, characterized in that The preparative liquid chromatography is a preparative liquid chromatography in a dynamic axial compression column mode; preferably, the inner diameter of the dynamic axial compression column is 400 to 1200 mm, and the packing height is 200 to 300 mm.

7. The method according to claim 6, characterized in that The filler of the dynamic axial compression column is octadecyl reverse phase silica gel, and preferably, the particle size of the silica gel is 8 to 12 μm.

8. The method according to any one of claims 1 to 7, characterized in that: The method further comprises: removing the solvent from the fractions collected by preparative liquid chromatography, re-crystallizing and drying to obtain the pure lanosterol.

9. The method according to claim 8, characterized in that The pressure of removing the solvent and / or the drying is -0.01 to -0.08 MPa, and the temperature is 40 to 45°C.

10. The method according to claim 8 or 9, characterized in that: The solvent used for the recrystallization is an alcohol solvent, preferably methanol. More preferably, the volume mass ratio of the alcohol solvent to the crude lanosterol is (5L-9L):1kg.