A method for preparing injectable cholesterol derived from egg yolk

Abstract

This invention relates to the field of pharmaceutical manufacturing, specifically to a method for preparing naturally sourced injectable cholesterol. The method includes the following steps: taking egg yolk oil, allowing it to settle, centrifuging the lower sediment at high speed, adding an organic solvent to the centrifuged solid for low-temperature extraction to remove residual glycerol lipids, taking the filtered extract, adding a first crystallization solvent for recrystallization and purification twice, taking the crude cholesterol product after recrystallization, adding a second crystallization solvent for further recrystallization and purification, and drying under reduced pressure to obtain the injectable cholesterol product. This invention solves the technical problem of preparing injectable cholesterol from egg yolk, is suitable for large-scale industrialization of cholesterol, and can be applied to the preparation of complex formulations such as liposomes, improving the safety of drug use.

Description

Technical Field

[0001] This invention relates to the field of natural drug extraction and separation, specifically to a method for preparing injectable cholesterol derived from egg yolk. Background Technology

[0002] Cholesterol, also known as cholesterol sterol, is a derivative of cyclopentanoperhydrophenanthrene. It is widely distributed in animal bodies, especially abundant in the brain and nerve tissue. Its solubility is similar to that of fats; it is insoluble in water but readily soluble in solvents such as ether and chloroform. In pharmaceutical excipients, it is mainly used as a emulsifier and emulsifier, and is an important excipient in novel targeted formulations such as liposomes and emulsions.

[0003] In the era of novel drug delivery systems, liposome injections, as a novel nano-drug delivery system, have attracted increasing attention in the pharmaceutical field due to their advantages such as good targeting and tissue affinity, improved solubility and penetration of poorly soluble drugs, and enhanced drug stability. Cholesterol is a basic auxiliary material in the manufacture of liposomes, playing a role in regulating the structure and properties of the membrane and greatly improving the carrying capacity and stability of liposomes. Therefore, greater attention is being paid to the source, content, impurity levels, and safety of cholesterol, especially in fields such as cell therapy and gene therapy.

[0004] Currently, cholesterol production mainly relies on extraction, with starting materials primarily derived from animal tissues such as pigs, cattle, and sheep, including viscera, brain marrow, and lanolin. Pharmaceutical-grade cholesterol has a purity of over 95%, with major impurities including β-cholesterol, sterols, 7-enylcholesterol, and 3β-hydroxy-5-en-4-methylcholesterol, all structurally similar to cholesterol. To meet the requirements for liposome injection preparation, cholesterol purity needs to be increased to over 98%, with impurities controlled at low levels to ensure formulation stability and safety. However, most domestic cholesterol manufacturers are limited by technological capabilities, making it difficult to achieve impurity control levels suitable for injection. Furthermore, increasing purity requires enhanced purification processes during cholesterol preparation, increasing production costs and reducing product yield, thus limiting the industrial production of high-purity cholesterol. In the field of synthetic chemistry, reported cholesterol chemical synthesis routes suffer from drawbacks such as relatively high raw material and catalyst prices, cumbersome process steps, highly toxic and polluting reagents, and low product purity, making them unsuitable for industrial production.

[0005] Patent CN 102617687 A, entitled "A Process for Preparing High-Purity Cholesterol," uses membrane separation technology and recrystallization to purify cholesterol from crude cholesterol, extracting cholesterol with a purity of over 95%. However, it does not test or evaluate other sterol impurities.

[0006] Patent CN 112110974 A, "A Method for Purifying Pharmaceutical Cholesterol", uses crude cholesterol as raw material and controls the intermediate steps of cholesterol purification through acid washing and alkali washing, thereby improving the cholesterol purity to ≥98.0%. It can control β-cholesterol to ≤0.6%, other individual impurities to ≤1.0%, and total impurities to ≤1.0%.

[0007] Patent CN 103102380 B, "A method for producing high-purity lanolin cholesterol," uses a mixed solvent of 95% ethanol and N-methylpyrrolidone to purify crude cholesterol, obtaining lanolin cholesterol with a purity of over 99%. Other sterol impurities were not detected or evaluated.

[0008] Patent CN 101817859 A, "Method for Separation and Extraction of Cholesterol from Lanolin Alcohol", describes a method for molecularly distilling lanolin alcohol, followed by recrystallization in a mixed solvent of methanol and acetone and then in an alcohol solvent to obtain a cholesterol product with a purity of over 90%.

[0009] Patent PCT / JP93 / 01823 describes a process where crude cholesterol is hydrogenated under the action of a catalyst to convert sterols in cholesterol into cholesterol. The cholesterol is then recrystallized to obtain cholesterol with a purity of over 99%, β-cholesterol ≤0.5%, and sterol ≤0.3%. However, the patent does not specify the exact recrystallization process.

[0010] The article "Study on a novel process for the separation of phospholipids, triacylglycerol and cholesterol from egg yolk" describes a method using ethanol at low temperature to remove most of the solidified triacylglycerol from the total lipids of egg yolk. Then, cholesterol was enriched from the remaining ethanol extract using β-cyclodextrin (β-CD), and further processed to obtain cholesterol.

[0011] Our company's patent CN 104177466 A, "A Preparation Process for Extracting Cholesterol from Egg Yolk Oil," uses egg yolk oil as the starting material. The process involves alumina column chromatography adsorption, followed by elution with a low-polarity solvent to remove low-polarity substances such as glycerides. Further elution with a mixed solvent yields crude cholesterol, which is then hydrolyzed and recrystallized to obtain high-purity cholesterol. However, analysis using the United States Pharmacopeia (USP) method revealed that the total amount of other sterols exceeded 0.5%, and β-cholesterol was >0.6%, failing to meet USP quality standards. Experiments show that the recrystallization step in the patented method cannot control the content of other sterols, while the column chromatography step can only effectively remove non-sterols. It is impossible to control the content of other sterols to meet USP quality standards by adjusting parameters such as the eluent combination, ratio, and dosage.

[0012] Based on the above experimental results, the column chromatography step in patent CN 104177466 A can only achieve efficient separation of non-sterol substances. However, due to the limitations of the adsorption material and the adsorption process principle used, the column chromatography step cannot effectively separate cholesterol from other structurally similar sterol substances. Furthermore, the recrystallization step in the patent cannot effectively separate cholesterol from other sterols.

[0013] Therefore, a new process is needed to solve the problem of effectively separating cholesterol from other structurally similar sterols, ensuring that the cholesterol content and other sterols meet the requirements of the European Pharmacopoeia and the United States Pharmacopoeia, and can be used for injection. Summary of the Invention

[0014] The purpose of this invention is to provide a method for preparing injectable cholesterol derived from egg yolk oil. The process is simple to operate and can be industrialized for large-scale production. It can not only ensure that the cholesterol content reaches more than 99%, but also ensure that other sterols meet the requirements of the European Pharmacopoeia and the United States Pharmacopoeia. Furthermore, it does not pose any safety hazards such as mad cow disease and has higher safety than animal-derived cholesterol on the market.

[0015] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0016] A method for preparing injectable cholesterol derived from egg yolk includes the following steps:

[0017] A. Take the egg yolk oil, let it stand and settle to obtain the upper clear liquid and the lower sediment, and then perform solid-liquid separation.

[0018] B. Take the lower sediment and centrifuge it to separate the solids after centrifugation;

[0019] C. Take the solid, add an organic solvent and stir. After solid-liquid separation, obtain a filter cake.

[0020] D. Take the filter cake, add the first crystallization solvent to dissolve it, cool it down to crystallize, filter it, discard the filter cake to obtain the filtrate, and concentrate the filtrate to recover the solvent to dryness to obtain the second concentrate;

[0021] E. Add the first crystallization solvent to the second concentrate to dissolve it, cool it down to crystallize, collect the crystals obtained from the second crystallization, and obtain crude cholesterol.

[0022] F. Take the crude cholesterol product, add the second crystallization solvent to dissolve it, cool it to crystallize, and dry it to obtain the cholesterol product for injection.

[0023] Egg yolk oil is collected and allowed to settle in a dark place. The lower sediment is then centrifuged at high speed. The centrifuged solid is extracted at low temperature with an organic solvent to remove residual glycerol lipids. The filtered extract is then recrystallized twice with a first crystallization solvent. The crude cholesterol product after recrystallization is then further refined with a second crystallization solvent. After vacuum drying, an injectable cholesterol product is obtained. This invention solves the technical problem of preparing injectable cholesterol from egg yolk, is suitable for large-scale industrialization of cholesterol, and can be applied to the preparation of complex formulations such as liposomes, improving the safety of drug use.

[0024] Currently, commercially available egg yolk oil is mainly produced through supercritical fluid extraction and solvent extraction. Both of these methods can be used as starting materials for cholesterol.

[0025] Preferably, in step C, the organic solvent comprises a mixture of alkanes and ketones; in steps D and E, the first crystallization solvent comprises a mixture of alcohols, ketones, and water; and in step F, the second crystallization solvent comprises alcohols and their aqueous solutions.

[0026] Preferably, in step C, the organic solvent comprises alkanes and ketones mixed in a volume ratio of 1:5 to 10; in steps D and E, the first crystallization solvent comprises alcohols, ketones, and water mixed in a volume ratio of 5 to 12:1:0.05 to 0.8; and in step F, the volume fraction of water in the second crystallization solvent is 0 to 5.0%.

[0027] Preferably, in step C, the alkane in the organic solvent includes one or more of n-hexane, petroleum ether, and cyclohexane, and the ketone includes one or more of acetone and butanone; in steps D and E, the alcohol in the first crystallization solvent includes one or more of methanol and ethanol, and the ketone includes one or more of acetone and butanone; in step F, the alcohol in the second crystallization solvent includes one or more of methanol and ethanol.

[0028] Preferably, in step C, the mass ratio of the organic solvent to the solid is 3 to 8:1; in step D, the mass ratio of the first crystallization solvent to the filter cake is 5 to 8:1; in step E, the mass ratio of the first crystallization solvent to the filter cake is 2 to 4:1; and in step F, the mass ratio of the second crystallization solvent to the crude cholesterol product is 1 to 3:1.

[0029] Preferably, in steps D and E, the cooling and crystallization temperature includes 0 to 35°C; in step F, the cooling and crystallization temperature includes 10 to 35°C.

[0030] Preferably, in step A, the static settling is carried out in a dark environment for a period of 30 days or more.

[0031] Preferably, in step A, the solid-liquid separation method includes filtration; in step B, centrifugation is performed in a high-speed centrifuge at a speed of 6000–15000 rpm; in step C, the solid-liquid separation includes filtration; and in step F, the drying includes vacuum drying.

[0032] Preferably, in step F, the temperature of the reduced pressure drying includes 50–70°C.

[0033] An injectable cholesterol product obtained according to the above-mentioned method for preparing injectable cholesterol derived from egg yolk.

[0034] Compared with the prior art, implementing the present invention has the following beneficial effects:

[0035] (1) The method of sedimentation enrichment-centrifugation separation of crude cholesterol in egg yolk oil can not only improve the cholesterol extraction efficiency and reduce the working time, but also obtain low cholesterol egg yolk oil without the introduction of organic solvents in the process, which can be used in food, cosmetics or pharmaceuticals.

[0036] (2) Crude cholesterol is refined using a multi-solvent system, and the cholesterol content of the finished product reaches more than 99%, while other sterols meet the requirements of the European Pharmacopoeia and the United States Pharmacopoeia.

[0037] (3) The process is simple to operate and can be industrialized for large-scale production. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments.

[0039] Example 1

[0040] Collect the egg yolk oil and let it stand in a dark place. Transfer the supernatant to a centrifuge, removing the lower precipitate. After centrifugation, crude cholesterol product 1 is obtained. Then, 50g of crude cholesterol product 1 is added to 150g of n-hexane:acetone (V:V = 1:8) and stirred at 35℃. The mixture is filtered, and the filter cake is dissolved in a mixed solvent of 250g of ethanol:water:butanone (V:V = 5.94:0.06:1) by heating. The mixture is then cooled to 35℃ to crystallize for 3 hours. After filtration, the solvent is recovered from the filtrate and dried. 200g of ethanol:water:butanone (V:V = 5.94:0.06:1) is added. The mixture was heated to dissolve the cholesterol, cooled to 35°C for 3 hours to crystallize, filtered, and the resulting filter cake was added to a 50g ethanol:water (V:V = 95:5) mixture and heated to dissolve. The mixture was then cooled to 10°C for recrystallization and purification for 4 hours. After drying under reduced pressure at 60°C for 8 hours, the final product, cholesterol, was obtained. The cholesterol content was 99.2%, β-cholestanol 0.08%, sterols 0.13%, enolanol 0.19%, 24-cholestipenol not detected, 3β-hydroxy-5-en-4-methylcholesterol 0.23%, and total sterol impurities 0.79%.

[0041] Example 2

[0042] Take the egg yolk oil and let it stand in a dark place. Transfer the lower precipitate and the upper clear liquid to a centrifuge. After centrifugation, crude cholesterol product 1 is obtained. Then, take 100g of crude cholesterol product 1 and add 500g of petroleum ether:acetone (V:V = 1:10). Stir at 15℃, filter, and add 800g of a mixed solvent of ethanol:water:acetone (V:V = 9.2:0.8:1) to the filter cake. Heat to dissolve, cool to 10℃ to crystallize for 6 hours, filter, recover the solvent from the filtrate to dryness, and add 300g of ethanol:water:acetone (V:V = 9.2... The mixture was dissolved in a 0.8:1) solvent by heating, cooled to 10°C for 6 hours to crystallize, filtered, and the resulting filter cake was dissolved in 200g of methanol by heating. The mixture was then cooled to 35°C for recrystallization and purification for 4 hours. After drying under reduced pressure at 70°C for 6 hours, the final product, cholesterol, was obtained. The cholesterol content was 99.3%, β-cholestanol 0.09%, sterols 0.12%, enolanol 0.23%, 24-cholestipenol not detected, 3β-hydroxy-5-en-4-methylcholesterol 0.18%, and total sterol impurities 0.62%.

[0043] Example 3

[0044] Collect the egg yolk oil and let it stand in a dark place. Transfer the lower precipitate and the upper clear liquid to a centrifuge. After centrifugation, crude cholesterol product 1 is obtained. Then, take 50g of crude cholesterol product 1 and add 300g of cyclohexane:butanone (V:V=1:5) and stir at 10℃. Filter, add 300g of methanol:water:acetone (V:V=11.4:0.6:1) mixed solvent to the filter cake and heat to dissolve. Cool to 20℃ to crystallize for 5 hours, filter, recover the solvent from the filtrate to dryness, and add 100g of methanol:water:acetone (V:V=11.4:0.6:1). The mixture was heated to dissolve the cholesterol, cooled to 20°C for 5 hours to crystallize, filtered, and the resulting filter cake was added to 150g of methanol:water (V:V = 97:3) and heated to dissolve. The mixture was then cooled to 10°C for recrystallization and purification for 5 hours. After drying under reduced pressure at 60°C for 8 hours, the final product, cholesterol, was obtained. The cholesterol content was 99.2%, β-cholestanol 0.13%, sterols 0.14%, enolanol 0.34%, 24-cholestipenol 0.03%, 3β-hydroxy-5-en-4-methylcholesterol 0.20%, and total sterol impurities 0.84%.

[0045] Example 4

[0046] Take the egg yolk oil and let it stand in a dark place. Transfer the supernatant to a centrifuge, removing the lower precipitate. After centrifugation, crude cholesterol product 1 is obtained. Then, 200g of crude cholesterol product 1 is added to 1200g of cyclohexane:acetone (V:V = 1:7) and stirred at 15℃. The mixture is filtered, and the filter cake is added to a mixed solvent of 1200g of ethanol:water:acetone (V:V = 9.9:0.1:1) and heated to dissolve. The mixture is then cooled to 15℃ to crystallize for 6 hours. After filtration, the solvent is recovered from the filtrate and dried. 400g of ethanol:water:acetone (V:V = 9.9...) is added... The mixture was dissolved in a 0.1:1) solvent by heating, cooled to 25°C for 5 hours to crystallize, filtered, and the resulting filter cake was dissolved in 200g of ethanol by heating. The mixture was then cooled to 15°C for recrystallization and purification for 6 hours. After drying under reduced pressure at 70°C for 6 hours, the final product, cholesterol, was obtained. The cholesterol content was 99.4%, β-cholestanol 0.10%, sterols 0.11%, enolanol 0.15%, 24-cholestipenol not detected, 3β-hydroxy-5-en-4-methylcholesterol 0.22%, and total sterol impurities 0.58%.

[0047] Example 5

[0048] Take the egg yolk oil and let it stand in a dark place. Transfer the lower precipitate and the upper clear liquid to a centrifuge. After centrifugation, crude cholesterol product 1 is obtained. Then, take 150g of crude cholesterol product 1 and add 1200g of n-hexane:butanone (V:V = 1:6) and stir at 10℃. Filter. Add 1200g of a mixed solvent of ethanol:water:butanone (V:V = 7.6:0.4:1) to the filter cake and heat to dissolve. Cool to 20℃ to crystallize for 5 hours. Filter, recover the solvent from the filtrate to dryness, and add 450g of a mixture of ethanol:water:butanone (V:V = 7.6:0.4:1). The mixture was dissolved in a solvent by heating, cooled to 20°C for 5 hours to crystallize, filtered, and the resulting filter cake was dissolved in a 150g ethanol:water (V:V = 95:5) mixed solvent by heating. The mixture was then cooled to 25°C for recrystallization and purification for 6 hours. After drying under reduced pressure at 60°C for 8 hours, the final product, cholesterol, was obtained. The cholesterol content was 99.3%, β-cholesterol 0.13%, sterol 0.12%, encholesterol 0.16%, 24-choledadienol not detected, 3β-hydroxy-5-en-4-methylcholesterol 0.21%, and total sterol impurities 0.62%.

[0049] Comparative Example 1

[0050] Take egg yolk oil and let it stand in a dark place. Transfer the supernatant to a centrifuge after removing the lower precipitate. Centrifuge to obtain crude cholesterol product 1. Take 50g of crude cholesterol product 1, 10g of medical activated charcoal, and 300mL of 97% ethanol solution. Heat the solution to boiling with stirring until the cholesterol is completely dissolved. Reflux for 60 minutes and filter at a constant temperature of 75℃. Cool the filtrate to room temperature with stirring and place it in an ice-water bath at 0℃. Continue stirring for 2 hours, then filter by suction. Wash the filter cake with cold 97% ethanol solution to obtain crude cholesterol product. Add the crude cholesterol product to 250mL of 97% ethanol solution and heat with stirring until boiling. Reflux for 60 minutes until the cholesterol is completely dissolved. Filter the solution through a 0.05m ceramic membrane at a constant temperature of 75℃ under conditions of a transmembrane pressure difference of 0.20MPa and a flow rate of 4.5m / s. After the filtrate was cooled to room temperature by stirring, it was placed in an ice-water bath at -5°C. Stirring was stopped after 2 hours, and the mixture was filtered. The filter cake was washed with cold 97% ethanol solution and dried to obtain a cholesterol sample. Analysis revealed the following contents: cholesterol 89.1%, β-cholestanol 0.40%, sterols 0.12%, enolanol 0.42%, 24-cholestipenol 0.08%, 3β-hydroxy-5-en-4-methylcholesterol 0.23%, and total sterol impurities 1.25%.

[0051] Comparative Example 2

[0052] Take egg yolk oil and let it stand in a dark place. Transfer the supernatant to a centrifuge after centrifugation to obtain crude cholesterol product 1. Take 50g of crude cholesterol product 1, add 1L of pharmaceutical ethanol, stir and dissolve at 78℃, reflux for 2 hours, adjust the pH to 4 with acid, cool at room temperature to crystallize, and filter. Dissolve the filter cake in 750mL of pharmaceutical ethanol at 76℃, reflux for 1.5 hours, adjust the pH to 7 with alkali, cool at room temperature to crystallize, then heat to dissolve, add 0.75g of activated carbon, stir at 70℃ for 10min, filter the adsorption solution while hot through a titanium rod, collect the filtrate and place it in a crystallization tank, cool at low temperature, crystallize, filter, and dry to obtain the cholesterol sample. The test results showed that the cholesterol content was 88.9%, β-cholestanol was 0.42%, sterol was 0.21%, encholestanol was 0.40%, 24-cholestipenol was 0.06%, 3β-hydroxy-5-en-4-methylcholesterol was 0.22%, and total sterol impurities were 1.31%.

[0053] Comparative Example 3

[0054] Egg yolk oil was collected and allowed to stand in a dark place. The lower precipitate and the upper clear liquid were transferred to a centrifuge. After centrifugation, crude cholesterol product 1 was obtained. 100g of crude cholesterol product 1 was then added to 300g of acetone and stirred at 10℃. After filtration, 50g of the filter cake was added to 750mL of 95% ethanol / N-methylpyrrolidone (V / V:8:1) and heated until completely dissolved. 0.28g of activated carbon was added and refluxed for 20 minutes. The mixture was then hot-filtered. After cooling and crystallization, the mixture was filtered and dried to obtain the cholesterol sample. The analysis showed that the cholesterol content was 92.1%, β-cholanol 0.40%, sterols 0.12%, enolol 0.40%, 24-cholestipenol 0.02%, 3β-hydroxy-5-en-4-methylcholesterol 0.20%, and total sterol impurities 1.14%.

[0055] Comparative Example 4

[0056] Take egg yolk oil and let it stand in a dark place. Transfer the lower precipitate and the upper clear liquid to a centrifuge. After centrifugation, crude cholesterol product 1 is obtained. Then, take 50g of crude cholesterol product 1 and heat it in 800g of a mixed solvent of methanol and acetone (V / V:1:1) until completely dissolved. Then, cool it to 10℃, keep it at this temperature for 8 hours, and then filter it under vacuum. Remove the solvent from the filtrate by vacuum evaporation to obtain a preliminary concentrate. Dissolve the preliminary concentrate in 8 times the volume of acetone by heating. Cool it to 18℃ and keep it at this temperature for 4 hours. Filter it under vacuum. Remove the solvent from the filtrate by vacuum evaporation to obtain a secondary concentrate. Dissolve the secondary concentrate in 7 times the volume of ethanol by heating. Cool it to 25℃ and keep it at this temperature for 8 hours. Filter it under vacuum to obtain crude cholesterol product. Recrystallize the crude cholesterol product in a methanol-acetone mixed solvent, filter it, and dry it to obtain a cholesterol sample. The test results showed that the cholesterol content was 94.5%, β-cholestanol 0.69%, sterol 0.10%, encholestanol 0.37%, 24-cholestipenol 0.03%, 3β-hydroxy-5-en-4-methylcholesterol 0.20%, and total sterol impurities 1.39%.

[0057] Comparative Example 5

[0058] Take the egg yolk oil and dissolve and stir it with cyclohexane as a solvent (dissolve 1L of n-hexane for every 1kg of crude egg yolk oil). After settling, the supernatant was passed through an alkaline alumina column (alumina to crude egg yolk oil weight ratio 5:1, column diameter ratio 1:3), and the remaining precipitate was added to the alumina column. The column was then eluted with cyclohexane (6 L of cyclohexane per 1 kg of alumina) to separate the egg yolk oil. The precipitate was then eluted with dichloromethane-anhydrous ethanol (volume ratio 1:3) (5 L of dichloromethane-anhydrous ethanol mixed solvent per 1 kg of alumina). The eluents were combined and concentrated to obtain crude cholesterol. Hydrolysis was performed in a 0.3 mol / L sodium hydroxide methanol solution (20 L of potassium hydroxide methanol solution per 1 kg of crude cholesterol). The methanol solution was adjusted to 95% (V / V) with appropriate amounts of hydrochloric acid, and the pH was controlled at 13. Crystallization was carried out at 10℃ for 60 min, filtered, and the filter cake was refluxed with methanol and cooled for crystallization once. 10 L of methanol (V / V) was added per 1 kg of crude cholesterol, and the mixture was dried to obtain refined cholesterol crystals. The test results showed that the cholesterol content was 97.5%, β-cholestanol 0.70%, sterol 0.20%, encholestanol 0.39%, 24-cholestipenol 0.03%, 3β-hydroxy-5-en-4-methylcholesterol 0.22%, and total sterol impurities 1.54%.

[0059] Comparative Example 6

[0060] Take the egg yolk oil and let it stand in a dark place. Transfer the lower precipitate and the upper clear liquid to a centrifuge. After centrifugation, crude cholesterol product 1 is obtained. Then, take 100g of crude cholesterol product 1 and add 500g of petroleum ether:acetone (V:V = 1:12). Stir at 15℃, filter, and add 800g of a mixed solvent of ethanol:water:acetone (V:V = 9.2:0.8:1) to the filter cake. Heat to dissolve, cool to 10℃ to crystallize for 6 hours, filter, recover the solvent from the filtrate to dryness, and add 300g of ethanol:water:acetone (V:V = 9.2... The mixture was dissolved in a 0.8:1) solvent by heating, cooled to 10°C for 6 hours to crystallize, filtered, and the resulting filter cake was dissolved in 200g of methanol by heating. The mixture was then cooled to 35°C for recrystallization and purification for 4 hours. After drying under reduced pressure at 70°C for 6 hours, the final product, cholesterol, was obtained. The cholesterol content was 97.4%, β-cholestanol 0.52%, sterols 0.08%, enoltanol 0.52%, 24-cholestipenol not detected, 3β-hydroxy-5-en-4-methylcholesterol 0.22%, and total sterol impurities 1.34%.

[0061] Comparative Example 7

[0062] Take the egg yolk oil and let it stand in a dark place. Transfer the lower precipitate and the upper clear liquid to a centrifuge. After centrifugation, crude cholesterol product 1 is obtained. Then, take 100g of crude cholesterol product 1 and add 500g of petroleum ether:acetone (V:V = 1:10) and stir at 15℃. Filter and add 800g of ethanol:water:acetone (V:V = 13.5:1.5:1) mixed solvent to the filter cake. Heat to dissolve and cool to 10℃ to crystallize for 6 hours. Filter and recover the solvent from the filtrate until dry. Add 300g of ethanol:water:acetone (V:V = 13.5:1.5:1) mixed solvent. The mixture was dissolved in a 5:1.5:1) solvent by heating, cooled to 10°C for 6 hours to crystallize, filtered, and the resulting filter cake was dissolved in 200g of methanol by heating. The mixture was then cooled to 35°C for recrystallization and purification for 4 hours. After drying under reduced pressure at 70°C for 6 hours, the final product, cholesterol, was obtained. The cholesterol content was 98.2%, β-cholestanol 0.70%, sterols 0.20%, enolanol 0.53%, 24-cholestipenol not detected, 3β-hydroxy-5-en-4-methylcholesterol 0.17%, and total sterol impurities 1.6%.

[0063] Comparative Example 8

[0064] Take the egg yolk oil and let it stand in a dark place. Transfer the lower precipitate and the upper clear liquid to a centrifuge. After centrifugation, crude cholesterol product 1 is obtained. Then, take 100g of crude cholesterol product 1 and add 500g of petroleum ether:acetone (V:V = 1:10) and stir at 15℃. Filter. Add 800g of a mixed solvent of ethanol:water:acetone (V:V = 10:0:1) to the filter cake and heat to dissolve. Cool to 10℃ to crystallize for 6 hours. Filter, recover the solvent from the filtrate to dryness, and add 300g of ethanol:water:acetone (V:V = 10:0:1) to the filtrate. The mixture was dissolved in a 0:1) solvent by heating, cooled to 10°C for 6 hours to crystallize, filtered, and the resulting filter cake was dissolved in 200g of methanol by heating. The mixture was then cooled to 35°C for recrystallization and purification for 4 hours. After drying under reduced pressure at 70°C for 6 hours, the final product, cholesterol, was obtained. The cholesterol content was 98.5%, β-cholestanol 0.68%, sterols 0.18%, enoltanol 0.52%, 24-cholestipenol not detected, 3β-hydroxy-5-en-4-methylcholesterol 0.19%, and total sterol impurities 1.57%.

[0065] Comparative Example 9

[0066] Take the egg yolk oil and let it stand in a dark place. Transfer the lower precipitate and the upper clear liquid to a centrifuge. After centrifugation, crude cholesterol product 1 is obtained. Then, take 100g of crude cholesterol product 1 and add 500g of petroleum ether:acetone (V:V = 1:10) and stir at 15℃. Filter. Add 800g of a mixed solvent of ethanol:water:acetone (V:V = 9.2:0.8:1) to the filter cake and heat to dissolve. Cool to 10℃ to crystallize for 6 hours. Filter, recover the solvent from the filtrate to dryness, and add 300g of a mixture of ethanol:water:acetone (V:V = 9.2:0.8:1). The solvent was heated to dissolve the product, then cooled to 10°C to crystallize for 6 hours. After filtration, the filter cake was added to 200g of methanol:water (V:V = 90:10) and heated to dissolve. The mixture was then cooled to 35°C for recrystallization and purification for 4 hours. Finally, the product was dried under reduced pressure at 70°C for 6 hours to obtain the final product, cholesterol, with a cholesterol content of 98.7%, β-cholestanol 0.33%, sterols 0.11%, enolanol 0.15%, 24-cholestipenol 0.07%, 3β-hydroxy-5-en-4-methylcholesterol 0.22%, and total sterol impurities of 0.88%.

[0067] Example of effect 1

[0068] The test results from the examples and comparative examples show that:

[0069] Compared to the examples, Comparative Examples 1-5 used conventional methods to extract cholesterol from egg yolk oil, resulting in lower cholesterol content and difficulty in meeting the requirements of the European Pharmacopoeia and the United States Pharmacopoeia for other sterols. Existing technical routes cannot meet the requirements for preparing high-purity injectable cholesterol from egg yolk. In Comparative Example 6, the proportion of ketones in the organic solvent was too high, resulting in a cholesterol purity level below 99%. In Comparative Example 7, the proportion of water in the first crystallization solvent was too high, and the proportion of acetone was too low, resulting in a cholesterol purity level below 99% and β-cholesterol exceeding the limit. In Comparative Example 8, the first crystallization solvent contained no water, resulting in a cholesterol purity level below 99% and β-cholesterol exceeding the limit. In Comparative Example 9, the proportion of water in the second crystallization solvent was too high, resulting in a cholesterol purity level below 99%. Only by using Examples 1-5 of the present invention can a high-purity (above 99%) cholesterol product be obtained, and the content of other sterols also meets the requirements of the European Pharmacopoeia and the United States Pharmacopoeia.

[0070] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, any equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A method for preparing injectable cholesterol derived from egg yolk, characterized in that, Includes the following steps: A. Take the egg yolk oil, let it stand and settle to obtain the upper clear liquid and the lower sediment, and then perform solid-liquid separation. B. Take the lower sediment and centrifuge it to separate the solids after centrifugation; C. Take the solid, add an organic solvent and stir. After solid-liquid separation, obtain a filter cake. D. Take the filter cake, add the first crystallization solvent to dissolve it, cool it down to crystallize, filter it, discard the filter cake to obtain the filtrate, and concentrate the filtrate to recover the solvent to dryness to obtain the second concentrate; E. Add the first crystallization solvent to the second concentrate to dissolve it, then cool it down to crystallize and obtain crude cholesterol. F. Take the crude cholesterol product, add the second crystallization solvent to dissolve it, cool it to crystallize, and dry it to obtain the cholesterol product for injection. In steps D and E, the first crystallization solvent comprises alcohols, ketones, and water mixed in a volume ratio of 5~12:1:0.05~0.8; in step F, the second crystallization solvent comprises alcohols and their aqueous solutions; in step F, the volume fraction of water in the second crystallization solvent is 0~5.0%; in steps D and E, the first crystallization solvent comprises one or more of methanol and ethanol, and one or more of acetone and butanone; in step F, the second crystallization solvent comprises one or more of methanol and ethanol.

2. The method for preparing injectable cholesterol derived from egg yolk according to claim 1, characterized in that, In step C, the organic solvent comprises a mixture of alkanes and ketones.

3. The method for preparing injectable cholesterol derived from egg yolk according to claim 2, characterized in that, In step C, the organic solvent comprises alkanes and ketones mixed in a volume ratio of 1:5 to 10.

4. The method for preparing injectable cholesterol derived from egg yolk according to claim 2, characterized in that, In step C, the organic solvent includes one or more of the following: alkanes: n-hexane, petroleum ether, and cyclohexane; and ketones: one or more of the following: acetone and butanone.

5. The method for preparing injectable cholesterol derived from egg yolk according to claim 1, characterized in that, In step C, the mass ratio of the organic solvent to the solid is 3~8:1; in step D, the mass ratio of the first crystallization solvent to the filter cake is 5~8:1; in step E, the mass ratio of the first crystallization solvent to the filter cake is 2~4:1; in step F, the mass ratio of the second crystallization solvent to the crude cholesterol product is 1~3:

1.

6. The method for preparing injectable cholesterol derived from egg yolk according to claim 1, characterized in that, In steps D and E, the cooling temperature for crystallization is 0~35℃; in step F, the cooling temperature for crystallization is 10~35℃.

7. The method for preparing injectable cholesterol derived from egg yolk according to claim 1, characterized in that, In step A, the static settling is carried out in a dark environment for a period of 30 days or more.

8. The method for preparing injectable cholesterol derived from egg yolk according to claim 1, characterized in that, In step A, the solid-liquid separation method includes filtration; in step B, centrifugation is carried out in a high-speed centrifuge at a speed of 6000~15000 rpm. In step C, the solid-liquid separation includes filtration; in step F, the drying includes vacuum drying.

9. The method for preparing injectable cholesterol derived from egg yolk according to claim 8, characterized in that, In step F, the temperature for vacuum drying is 50~70℃.