A method for preparing a natural fish oil-derived injectable cholesterol

Abstract

The present application relates to the field of medicine manufacturing, and relates to a preparation method of natural source cholesterols for injection, which comprises the following steps: taking fish oil, cooling and winterizing, taking the lower sediment, heating to dissolve it into clear winterization fraction, adding the winterization fraction into a chromatographic column filled with mixed filler of adsorbent and filter aid which are mixed uniformly in a physical way to perform circulation chromatography, emptying the winterization fraction after the circulation ends, adding eluent to the chromatographic column, and recovering the solvent after the eluent is combined to obtain cholesterols concentrate. The cholesterols concentrate is added with first crystallization solvent to recrystallize and refine twice, the cholesterols crude product after recrystallization is taken and added with second crystallization solvent to recrystallize and refine, and the product is dried under reduced pressure to obtain cholesterols for injection. The present application solves the technical problem of preparing cholesterols for injection from fish oil, is suitable for large-scale industrialization of cholesterols, can be applied to preparation of complex preparations such as liposomes, and improves the safety of drug use.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical manufacturing, specifically to a method for preparing injectable cholesterol derived from natural fish oil. 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. Cholesterol is the most abundant sterol compound in animals. It is a component of cell membranes and a precursor to steroid hormones, bile acids, and vitamin D. As a pharmaceutical excipient, cholesterol primarily serves as an emulsifier and a carrier material for liposome inclusions.

[0003] With the continuous development of new drug delivery systems, 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. Therefore, the European Pharmacopoeia and the United States Pharmacopoeia have stricter quality requirements for pharmaceutical-grade cholesterol. Currently, cholesterol production is mainly based on extraction. The starting materials for extraction primarily come from animal tissues such as pigs, cattle, and sheep, including offal and brain marrow, as well as lanolin. Extraction can also be obtained from poultry eggs and egg products, and marine biological byproducts.

[0004] 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.

[0005] Traditional methods for obtaining cholesterol from lanolin are complex and costly, and some cholesterol does not meet the purity requirements for injection.

[0006] 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 adsorption by alumina column chromatography, elution with a low-polarity solvent to remove low-polarity substances such as glycerides, and further elution with a mixed solvent to obtain crude cholesterol. The crude cholesterol is then subjected to hydrolysis and recrystallization to obtain high-purity cholesterol.

[0007] The process of obtaining cholesterol from egg yolks is relatively simple, but the purity of the cholesterol does not meet the requirements for injection grade.

[0008] In addition to the sources mentioned above, research on extracting cholesterol from fish oil has been increasing in recent years. Fish oil mainly contains about 1-3% cholesterol, and the cholesterol content can be increased to 5-50% through enrichment. Therefore, cholesterol can be extracted from fish oil, expanding the range of raw materials for cholesterol production and achieving efficient utilization of fish oil resources. To this end, there are reports in China on separating cholesterol from fish oil; however, most of the reported methods use saponification reactions, which result in unstable processes, high reaction temperatures, high solvent consumption, high production costs, and a large amount of waste generated, causing significant environmental pollution and hindering industrial production.

[0009] For example, patent CN 111315855 B, "An Improved Method for Extracting Cholesterol from Fish Oil Residue", uses 4-dimethylaminopyridine as a catalyst to saponify fish oil residue. After neutralization, phase separation, inorganic salt addition, cholesterol separation by adduct, and solvent crystallization, cholesterol with a purity of 95-97% is obtained.

[0010] Patent CN 116693588 A, "A Method for Extracting High-Purity Cholesterol from Fish Oil Residue," describes a method for extracting high-purity cholesterol from fish oil residue through saponification, calcium salt complexation, organic solvent extraction, acid-base adjustment, and recrystallization, resulting in cholesterol with a purity of over 95%.

[0011] Patent CN 109705184A, "A Continuous Method for Extracting High-Purity Cholesterol from Fish Oil Residue," describes a method for saponifying fish oil residue using an aqueous solution of potassium hydroxide (or sodium hydroxide). The saponified solution is then countercurrently extracted using a mixed solvent of n-heptane and ethyl acetate to obtain an extract. The extract is then countercurrently washed with a mixed solvent of water, methanol, and acetic acid. The solvent is recovered by distillation after washing, and the residue is recrystallized with ethanol to obtain high-purity cholesterol.

[0012] Furthermore, cholesterol derived from fish oil faces the same quality control challenges as cholesterol samples extracted from traditional sources. Sterol impurities in cholesterol, such as β-cholesterol, sterols, 7-enylcholesterol, and 3β-hydroxy-5-en-4-methylcholesterol, have structures similar to cholesterol, making them difficult to effectively control and ensuring that the cholesterol product content and other sterols meet the requirements of the European Pharmacopoeia and the United States Pharmacopoeia, thus failing to meet the quality requirements of pharmaceutical production. Therefore, to meet the needs of preparing novel injectable formulations, it is necessary to expand the sources of cholesterol raw materials, improve cholesterol purity, control impurities at low levels, ensure the stability and safety of the formulation, and provide high-quality, low-cost cholesterol excipients for the development of new formulations. Summary of the Invention

[0013] The purpose of this invention is to provide a method for preparing injectable cholesterol from natural fish oil, which solves the technical problem of preparing injectable cholesterol from fish oil, is suitable for large-scale industrialization of cholesterol, can be applied to the preparation of complex formulations such as liposomes, and improves the safety of drug use.

[0014] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0015] A method for preparing injectable cholesterol from natural fish oil includes the following steps:

[0016] A. Take fish oil, cool it down for winterization, obtain the upper clear liquid and the lower sediment, and perform solid-liquid separation;

[0017] B. Take the lower sediment and heat it to dissolve it to obtain the winterized fraction;

[0018] C. Take the winterized fraction and use a chromatography column filled with mixed packing material as the stationary phase for cyclic chromatography;

[0019] D. After the cyclic chromatography is completed, eluent is added to the chromatography column to elute, the eluent is collected, concentrated, and cholesterol concentrate is obtained.

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

[0021] F. 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] G. 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] This invention relates to the field of pharmaceutical manufacturing, specifically to a method for preparing naturally sourced injectable cholesterol, comprising the following steps: taking fish oil, cooling and winterizing it, taking the lower sediment and heating it to dissolve it into a clear winterized fraction, adding the winterized fraction to a chromatography column packed with a physically mixed adsorbent and filter aid for cyclic chromatography, purging the winterized fraction after circulation, adding eluent to the chromatography column, combining the eluents and recovering the solvent to obtain a cholesterol concentrate. The cholesterol concentrate is recrystallized twice with a first crystallization solvent, and the crude cholesterol product after recrystallization is then recrystallized again with a second crystallization solvent, and dried under reduced pressure to obtain the injectable cholesterol product.

[0024] In step A, the purpose of winterization of fish oil is to enrich cholesterol in the lower sediment. After winterization, the fish oil can continue to be used for the preparation of refined fish oil. Taking the sediment for cholesterol preparation can improve the cholesterol enrichment efficiency in step C.

[0025] This invention solves the technical problem of preparing injectable cholesterol from fish oil, making it suitable for large-scale industrialization of cholesterol. It can be applied to the preparation of complex formulations such as liposomes, thus improving the safety of drug use.

[0026] Preferably, in step D, the eluent comprises a mixture of alkanes, alcohols, and water; in steps E and F, the first crystallization solvent comprises a mixture of alcohols, ketones, and water; and in step G, the second crystallization solvent comprises alcohols and their aqueous solutions.

[0027] Preferably, in step D, the eluent comprises alkanes, alcohols, and water mixed in a volume ratio of 1:1 to 5:0.0001 to 0.0015; in steps E and F, the first crystallization solvent comprises alcohols, ketones, and water mixed in a volume ratio of 3 to 10:1:0.06 to 0.8; and in step G, the volume fraction of water in the second crystallization solvent is 0 to 8.0%.

[0028] Preferably, in step D, the eluent contains one or more alkanes including n-hexane, petroleum ether, cyclohexane, dichloromethane, and chloroform, and alcohols including one or more methanol, ethanol, and isopropanol; in steps E and F, the first crystallization solvent contains one or more alcohols including methanol, ethanol, and benzyl alcohol, and ketones including one or more acetone, butanone, and methyl isobutyl ketone; in step F, the second crystallization solvent contains one or more alcohols including methanol and ethanol.

[0029] Preferably, in step D, the mass ratio of the eluent to the mixed packing material is 3 to 8:1; in step E, the mass ratio of the first crystallization solvent to the cholesterol concentrate is 4 to 7:1; in step F, the mass ratio of the first crystallization solvent to the cholesterol concentrate is 1 to 3:1; in step F, the mass ratio of the second crystallization solvent to the crude cholesterol product is 1 to 3:1.

[0030] Preferably, in step A, the temperature for the winterization cooling includes -10 to 0°C; in step B, the temperature for the heating and dissolution of the lower sediment includes 20 to 50°C to obtain the winterization fraction; in step C, the mixed packing material includes a mixture of adsorbent and filter aid; the mass ratio of the adsorbent to the winterization fraction includes 0.05 to 0.3:1; and the mass ratio of the filter aid to the winterization fraction includes 0.1 to 0.5:1.

[0031] Preferably, in step C, the mixed packing material comprises an adsorbent and a filter aid that are uniformly mixed in a physical manner; the adsorbent comprises one or more of alumina, silica gel, and aminosilica gel; and the filter aid comprises one or more of diatomaceous earth, bentonite, and kaolin.

[0032] In this invention, a filter aid is used to reduce resistance during the circulating column chromatography process, so as to prevent the flow rate from being too slow or the chromatography column from becoming clogged.

[0033] Preferably, in step C, the specific conditions for the cyclic chromatography include: temperature 30–50°C, pressure 0.01–1.0 MPa, and contact time 2–10 h; the winterization fraction is circulated into the chromatography column 1–5 times.

[0034] Preferably, in step A, the solid-liquid separation method includes filtration; in steps E and F, the cooling crystallization temperature includes -10 to 25°C; in step G, the cooling crystallization temperature includes -10 to 25°C; and in step G, the drying includes vacuum drying.

[0035] Preferably, the temperature for vacuum drying includes 50–70°C.

[0036] An injectable cholesterol product obtained by the above-mentioned method for preparing injectable cholesterol derived from natural fish oil.

[0037] An injectable cholesterol product obtained according to the above-mentioned preparation method of injectable cholesterol from natural fish oil.

[0038] Compared with the prior art, implementing the present invention has the following beneficial effects:

[0039] (1) The method of winterization enrichment of crude cholesterol in fish oil can not only improve the efficiency of subsequent chromatographic cholesterol enrichment and separation and reduce working time, but also allow the winterized low-cholesterol fish oil to continue to be used in food, cosmetics or medicines, thus improving the utilization rate of fish oil.

[0040] (2) The use of cyclic chromatography elution technology can effectively enrich sterols in fish oil, effectively remove low polar substances such as glycerides, improve the efficiency of cholesterol refining and purification, save material and labor costs, reduce energy consumption and reduce environmental pressure compared with traditional saponification methods.

[0041] (3) 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.

[0042] (4) The process is simple to operate and can be industrialized for large-scale production. Detailed Implementation

[0043] 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.

[0044] Example 1

[0045] Fish oil was winterized with solvent. The lower sediment was heated at 30°C to dissolve it into a clear winterized fraction. 300g of alumina and 50g of diatomaceous earth were weighed, physically mixed thoroughly, and added to a chromatography column. 1kg of the winterized fraction was subjected to the above-described mixed-packing column chromatography operation, with the column temperature controlled at 35±2°C, the pressure controlled at 0.15±0.02mPa, and the contact time at 8h. After circulation, the winterized fraction was purged, and 1750ml of n-hexane:methanol:water (V:V:V = 20:80:0.05) was added to the chromatography column for elution. The eluents were combined, and the solvent was recovered to obtain a cholesterol concentrate. Cholesterol concentrate was dissolved in a mixed solvent of ethanol:water:acetone (V:V = 5.94:0.06:1) by heating (6 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The solution was cooled to 10°C and crystallized over 4 hours. The mixture was filtered, and the solvent was recovered from the filtrate until dry. Then, another 2 kg of mixed solvent of ethanol:water:acetone (V:V = 5.94:0.06:1) was added and heated to dissolve the concentrate. The solution was cooled to 10°C and crystallized over 4 hours. The mixture was filtered, and the solvent was recovered from the filtrate until dry. The filter cake was dissolved by heating in a mixed solvent of ethanol:water (V:V = 97:3) (1 kg of mixed solvent was added for every 1 kg of filter cake), cooled to 5°C, recrystallized and purified for 5 h, and then dried under reduced pressure at 60°C for 8 h to obtain the final product cholesterol, with a cholesterol content of 99.2%, β-cholesterol 0.23%, sterol 0.13%, encholesterol 0.19%, 24-cholestipenol 0.03%, 3β-hydroxy-5-en-4-methylcholesterol 0.20%, and total sterol impurities of 0.78%.

[0046] Example 2

[0047] Fish oil was winterized with solvent. The lower sediment was heated at 25°C to dissolve it into a clear winterized fraction. 800g of silica gel and 200g of diatomaceous earth were weighed, physically mixed thoroughly, and added to a chromatography column. 2kg of the winterized fraction was subjected to the above-described mixed-packing column chromatography operation, with the column temperature controlled at 40±2°C, the pressure controlled at 0.20±0.02mPa, and the contact time at 6h. After circulation, the winterized fraction was purged, and 4000ml of cyclohexane:ethanol:water (V:V:V = 25:75:0.05) was added to the chromatography column for elution. The eluents were combined, and the solvent was recovered to obtain a cholesterol concentrate. Cholesterol concentrate was dissolved in a methanol:water:methyl ethyl ketone (V:V = 9.8:0.2:1) mixture and heated (7 kg of the mixture was added for every 1 kg of cholesterol concentrate). The solution was cooled to -10°C for 8 hours to allow crystals to crystallize. The mixture was filtered, and the solvent was recovered from the filtrate until dry. Then, another methanol:water:methyl ethyl ketone (V:V = 9.8:0.2:1) mixture was added and heated to dissolve the concentrate (1 kg of the mixture was added for every 1 kg of cholesterol concentrate). The solution was cooled to -10°C for 5 hours to allow crystals to crystallize. The mixture was filtered, and the solvent was recovered from the filtrate. The solution was dried to dryness, and the resulting filter cake was dissolved in anhydrous ethanol by heating (2 kg solvent per 1 kg filter cake). The solution was then cooled to 20°C and recrystallized for 4 hours. After drying under reduced pressure at 70°C for 6 hours, the final product, cholesterol, was obtained. The cholesterol content was 99.0%, β-cholesterol 0.29%, sterol 0.20%, encholesterol 0.21%, 24-cholestipenol 0.06%, 3β-hydroxy-5-en-4-methylcholesterol 0.20%, and total sterol impurities 0.96%.

[0048] Example 3

[0049] Fish oil was winterized with solvent. The lower sediment was heated at 25°C to dissolve it into a clear winterized fraction. 500g of alumina and 300g of bentonite were weighed, physically mixed thoroughly, and added to a chromatography column. 1kg of the winterized fraction was subjected to the above-described mixed-packing column chromatography operation, with the column temperature controlled at 30±2°C, the pressure controlled at 0.30±0.02mPa, and the contact time at 10h. After circulation, the winterized fraction was purged, and 6400ml of a mixture of chloroform:isopropanol:water (V:V:V = 25:75:0.1) was added to the column for elution. The eluents were combined, and the solvent was recovered to obtain a cholesterol concentrate. Cholesterol concentrate was dissolved in a mixed solvent of ethanol:water:acetone (V:V = 7.6:0.4:1) by heating (4 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The solution was cooled to 25°C and crystallized over 4 hours. The mixture was filtered, and the solvent was recovered from the filtrate until dry. Then, another mixed solvent of ethanol:water:acetone (V:V = 7.6:0.4:1) was added and heated to dissolve the concentrate (3 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The solution was cooled to 25°C and crystallized over 4 hours. The mixture was filtered, and the solvent was recovered from the filtrate until dry. The resulting filtrate... The filter cake was dissolved in a methanol:water (V:V = 98:2) mixture by heating (3 kg of solvent was added for every 1 kg of filter cake). The mixture was then cooled to -10℃ for recrystallization and purification for 8 hours. After drying under reduced pressure at 60℃ for 8 hours, the final product, cholesterol, was obtained. The cholesterol content was 99.2%, β-cholesterol 0.32%, sterol 0.11%, encholesterol 0.38%, 24-cholestipenol 0.05%, 3β-hydroxy-5-en-4-methylcholesterol 0.22%, and total sterol impurities 0.83%.

[0050] Example 4

[0051] Fish oil was winterized with solvent. The lower sediment was heated at 35°C to dissolve it into a clear winterized fraction. 150g of aminosilicone and 150g of kaolin were weighed, physically mixed evenly, and added to a chromatography column. 1.5kg of the winterized fraction was subjected to the above-described mixed-packing column chromatography operation. The column temperature was controlled at 35±2°C, the pressure at 0.15±0.02mPa, and the contact time at 10h. After circulation, the winterized fraction was purged, and 900ml of dichloromethane:ethanol:water (V:V:V = 50:50:0.01) was added to the chromatography column for elution. The eluents were combined, and the solvent was recovered to obtain a cholesterol concentrate. Cholesterol concentrate was dissolved in a mixed solvent of benzyl alcohol:water:methyl isobutyl ketone (V:V = 3.8:0.2:1) by heating (5 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The solution was cooled to 20°C and crystallized for 5 hours. The mixture was filtered, and the solvent was recovered from the filtrate until dry. Then, another mixed solvent of benzyl alcohol:water:methyl isobutyl ketone (V:V = 3.8:0.2:1) was added and dissolved in the solution by heating (2 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The solution was cooled to 20°C and crystallized for 5 hours. The mixture was filtered, and the solvent was recovered from the filtrate until dry. The obtained filter cake was dissolved in a mixed solvent of ethanol:water (V:V = 92:8) by heating (1 kg of solvent was added for every 1 kg of filter cake). The mixture was then cooled to 25°C and recrystallized for 5 hours. After drying under reduced pressure at 70°C for 5 hours, the final product, cholesterol, was obtained. The cholesterol content was 99.3%, β-cholestanol 0.13%, sterol 0.09%, encholestanol 0.33%, 24-cholestipenol 0.02%, 3β-hydroxy-5-en-4-methylcholesterol 0.14%, and total sterol impurities 0.71%.

[0052] Example 5

[0053] Fish oil was winterized with solvent. The lower sediment was heated at 35°C to dissolve it into a clear winterized fraction. 200g of aminosilicone and 200g of diatomaceous earth were weighed, physically mixed thoroughly, and added to a chromatography column. 1kg of the winterized fraction was subjected to the above-described mixed-packing column chromatography operation, with the column temperature controlled at 45±2°C, the pressure controlled at 0.20±0.02mPa, and the contact time at 10h. After circulation, the winterized fraction was purged, and 2400ml of petroleum ether:methanol:water (V:V:V = 33:67:0.15) was added to the chromatography column for elution. The eluents were combined, and the solvent was recovered to obtain a cholesterol concentrate. Cholesterol concentrate was dissolved in a methanol:water:methyl isobutyl ketone (V:V = 9.2:0.8:1) mixture by heating (5 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The mixture was cooled to 0°C and crystallized for 4 hours. After filtration, the solvent was recovered from the filtrate and dried. The resulting filter cake was dissolved in a methanol:water:methyl isobutyl ketone (V:V = 9.2:0.8:1) mixture by heating (1 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The mixture was cooled to 0°C and crystallized for 4 hours. After filtration, the solvent was recovered from the filtrate and dried. The resulting filter cake was dissolved in a methanol:water (V:V = 95:5) mixture by heating (2 kg of solvent was added for every 1 kg of filter cake). The mixture was cooled to 0°C and recrystallized for 4 hours. After drying under reduced pressure at 50°C for 8 hours, the final product, cholesterol, was obtained. The test results showed that the cholesterol content was 99.1%, β-cholestanol 0.16%, sterol 0.11%, encholestanol 0.43%, 24-cholestipenol 0.06%, 3β-hydroxy-5-en-4-methylcholesterol 0.17%, and total sterol impurities 0.93%.

[0054] Comparative Example 1

[0055] Add 400g of fish oil waste to 400ml of methanol. Add 40g of sodium hydroxide and 1g of 4-dimethylaminopyridine, and reflux for 1 hour. After 1 hour, distill the methanol at 70-75℃. At 75℃, add 600ml of 25% sulfuric acid and 300ml of water. Add 600ml of 2-butanone, and stir at 75℃ for 30 minutes. Separate the organic and aqueous layers. Wash the organic layer three times with 100ml of saturated salt solution. Evaporate the 2-butanone at 90-95℃. Add 800ml of 2-butanone to the above reaction mixture, and cool to 65-70℃. Add 190g of calcium bromide, and heat at 65-70℃ for 8 hours. Cool the reaction mixture to 30℃, filter, and wash with 100ml of 2-butanone. Suspend the solid residue in 600ml of methanol and reflux for 3 hours. The reaction mixture was cooled to 35°C and maintained at the same temperature for 3 hours. The mixture was filtered and washed with 150 ml of methanol. The solid residue was added to 1080 ml of toluene, heated to 45-50°C, filtered, and washed with 100 ml of toluene. The solvent was recovered from the filtrate and dried to obtain the residue. 400 ml of methanol was added to the residue and heated to 65°C, maintaining the temperature for 2 hours. The reaction mixture was cooled to 30-35°C and maintained for 2 hours. The solid was filtered and washed with 100 ml of methanol. The separated solid was dried under vacuum at 65-70°C to obtain the final product, cholesterol. Analysis showed that the cholesterol content was 96.7%, β-cholesterol 0.72%, sterols 0.27%, encholesterol 0.38%, 24-cholestipenol 0.13%, 3β-hydroxy-5-en-4-methylcholesterol 0.25%, and total sterol impurities 1.75%.

[0056] Comparative Example 2

[0057] Take 100g of fish oil and 100g of 20% sodium hydroxide aqueous solution, and stir at 70-80℃ for 3 hours to obtain a saponified solution. Cool the saponified solution to room temperature, add 100g of 20% calcium chloride aqueous solution dropwise, stir for 30 minutes, and filter to obtain a cholesterol calcium salt complex. Place the cholesterol calcium salt complex in a Soxhlet extractor, add 306g of acetone, and extract continuously 8 times at 80℃. Filter, and recover the solvent from the filtrate under reduced pressure to dryness to obtain a cholesterol concentrate. Dissolve the cholesterol concentrate in 50g of acetone, adjust the pH of the system to approximately 2-3 with concentrated hydrochloric acid, stir at room temperature for 3 hours, and filter to obtain crude cholesterol. Crude cholesterol was taken, and 35 mL of ethanol was added. The mixture was heated to 70–80 °C and stirred for 2 hours. It was then filtered while hot. The filtrate was cooled to 0 °C, kept warm and stirred to allow crystallization for 1 hour, and then filtered again to obtain a first-stage purified cholesterol sample. Another 30 mL of ethanol was added to the first-stage purified sample, and the mixture was heated to 70–80 °C until all the solids dissolved. The mixture was then cooled to 0 °C to allow crystallization for 1 hour, filtered, and dried to obtain the final product, cholesterol. Analysis showed that the cholesterol content was 95.1%, β-cholanol 0.74%, sterols 0.39%, encholanol 0.33%, 24-cholestipenol 0.22%, 3β-hydroxy-5-en-4-methylcholesterol 0.26%, and total sterol impurities 1.94%.

[0058] Comparative Example 3

[0059] Weigh 3000g of fish oil residue, add 1200mL of 7mol / L potassium hydroxide aqueous solution, heat under reflux for 1h, and cool to room temperature to obtain the saponification solution. Use 1200ml of n-heptane and ethyl acetate (V:V = 1:1) as the extraction solvent for extraction three times. Combine the extracts and mix with methanol-water (V:V = 1:3) solvent. Adjust the pH to 3.7 with acetic acid as the washing agent, washing three times with 1000ml each time. Distill the cholesterol extract under reduced pressure to recover the solvent. Recrystallize the residue with ethanol and dry to obtain the final product, cholesterol. Analysis showed a cholesterol content of 98.2%, β-cholesterol 0.68%, sterols 0.25%, encholesterol 0.40%, 24-cholestipenol 0.19%, 3β-hydroxy-5-en-4-methylcholesterol 0.25%, and total sterol impurities of 1.77%.

[0060] Comparative Example 4

[0061] Take the fish oil and dissolve it in cyclohexane as a solvent by stirring (1L of n-hexane is used to dissolve 1kg of fish 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. Elution was continued with cyclohexane (6 L of cyclohexane per 1 kg of alumina) to separate the glycerides. Elution was then carried out 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. After filtration, the filter cake was refluxed with methanol and cooled for crystallization once, with 10 L of methanol (V / V) added per 1 kg of crude cholesterol. The mixture was dried to obtain refined cholesterol crystals. The test results showed that the cholesterol content was 98.2%, β-cholestanol 0.64%, sterol 0.22%, encholestanol 0.41%, 24-cholestipenol 0.03%, 3β-hydroxy-5-en-4-methylcholesterol 0.22%, and total sterol impurities 1.52%.

[0062] Comparative Example 5

[0063] Fish oil was winterized with solvent. The lower sediment was heated at 35°C to dissolve it into a clear winterized fraction. 400g of activated clay was weighed, physically mixed thoroughly, and added to a chromatography column. 1kg of the winterized fraction was subjected to the above-described mixed-packing column chromatography operation, with the column temperature controlled at 45±2°C, the pressure at 0.20±0.02mPa, and the contact time at 10h. After circulation, the winterized fraction was purged, and 2400ml of petroleum ether:methanol:water (V:V:V = 33:67:0.15) was added to the column for elution. The eluents were combined, and the solvent was recovered to obtain a cholesterol concentrate. Cholesterol concentrate was dissolved in a methanol:water:methyl isobutyl ketone (V:V = 9.2:0.8:1) mixture by heating (5 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The mixture was cooled to 0°C and crystallized for 4 hours. After filtration, the solvent was recovered from the filtrate and dried. The resulting filter cake was dissolved in a methanol:water:methyl isobutyl ketone (V:V = 9.2:0.8:1) mixture by heating (1 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The mixture was cooled to 0°C and crystallized for 4 hours. After filtration, the solvent was recovered from the filtrate and dried. The resulting filter cake was dissolved in a methanol:water (V:V = 95:5) mixture by heating (2 kg of solvent was added for every 1 kg of filter cake). The mixture was cooled to 0°C and recrystallized for 4 hours. After drying under reduced pressure at 50°C for 8 hours, the final product, cholesterol, was obtained. The test results showed that the cholesterol content was 95.6%, β-cholestanol 0.65%, sterol 0.23%, encholestanol 0.43%, 24-cholestipenol 0.08%, 3β-hydroxy-5-en-4-methylcholesterol 0.20%, and total sterol impurities 1.59%.

[0064] Comparative Example 6

[0065] Fish oil was winterized with solvent. The lower sediment was heated at 35°C to dissolve it into a clear winterized fraction. 200g of aminosilicone and 200g of diatomaceous earth were weighed, physically mixed thoroughly, and added to a chromatography column. 1kg of the winterized fraction was then subjected to the above-described mixed-packing column chromatography operation. The column temperature was controlled at 45±2°C, the pressure at 0.20±0.02mPa, and the contact time at 10h. After circulation, the winterized fraction was purged, and 2400ml of petroleum ether:methanol:water (V:V:V = 14.3:85.7:0.2) was added to the column for elution. The eluents were combined, and the solvent was recovered to obtain a cholesterol concentrate. Cholesterol concentrate was dissolved in a methanol:water:methyl isobutyl ketone (V:V = 9.2:0.8:1) mixture by heating (5 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The mixture was cooled to 0°C and crystallized for 4 hours. After filtration, the solvent was recovered from the filtrate and dried. The resulting filter cake was dissolved in a methanol:water:methyl isobutyl ketone (V:V = 9.2:0.8:1) mixture by heating (1 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The mixture was cooled to 0°C and crystallized for 4 hours. After filtration, the solvent was recovered from the filtrate and dried. The resulting filter cake was dissolved in a methanol:water (V:V = 95:5) mixture by heating (2 kg of solvent was added for every 1 kg of filter cake). The mixture was cooled to 0°C and recrystallized for 4 hours. After drying under reduced pressure at 50°C for 8 hours, the final product, cholesterol, was obtained. The test results showed that the cholesterol content was 97.3%, β-cholestanol was 0.69%, sterol was 0.21%, encholestanol was 0.51%, 24-cholestipenol was 0.13%, 3β-hydroxy-5-en-4-methylcholesterol was 0.20%, and total sterol impurities were 1.74%.

[0066] Comparative Example 7

[0067] Fish oil was winterized with solvent. The lower sediment was heated at 35°C to dissolve it into a clear winterized fraction. 200g of aminosilicone and 200g of diatomaceous earth were weighed, physically mixed thoroughly, and added to a chromatography column. 1kg of the winterized fraction was subjected to the above-described mixed-packing column chromatography operation. The column temperature was controlled at 45±2°C, the pressure at 0.20±0.02mPa, and the contact time at 10h. After circulation, the winterized fraction was purged, and 2400ml of petroleum ether:methanol:water (V:V:V = 33:67:0.15) was added to the column for elution. The eluents were combined, and the solvent was recovered to obtain a cholesterol concentrate. Cholesterol concentrate was dissolved in a methanol:water:methyl isobutyl ketone (V:V = 10.8:1.2:1) mixture by heating (5 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The mixture was cooled to 0°C and crystallized for 4 hours. After filtration, the solvent was recovered from the filtrate and dried. The resulting filter cake was dissolved in a methanol:water:methyl isobutyl ketone (V:V = 10.8:1.2:1) mixture by heating (1 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The mixture was cooled to 0°C and crystallized for 4 hours. After filtration, the solvent was recovered from the filtrate and dried. The resulting filter cake was dissolved in a methanol:water (V:V = 95:5) mixture by heating (2 kg of solvent was added for every 1 kg of filter cake). The mixture was cooled to 0°C and recrystallized for 4 hours. After drying under reduced pressure at 50°C for 8 hours, the final product, cholesterol, was obtained. The test results showed that the cholesterol content was 98.4%, β-cholestanol was 0.70%, sterol was 0.20%, encholestanol was 0.22%, 24-cholestipenol was not detected, 3β-hydroxy-5-en-4-methylcholesterol was 0.16%, and total sterol impurities were 1.28%.

[0068] Comparative Example 8

[0069] Fish oil was winterized with solvent. The lower sediment was heated at 35°C to dissolve it into a clear winterized fraction. 200g of aminosilicone and 200g of diatomaceous earth were weighed, physically mixed thoroughly, and added to a chromatography column. 1kg of the winterized fraction was subjected to the above-described mixed-packing column chromatography operation. The column temperature was controlled at 45±2°C, the pressure at 0.20±0.02mPa, and the contact time at 10h. After circulation, the winterized fraction was purged, and 2400ml of petroleum ether:methanol:water (V:V:V = 33:67:0.15) was added to the column for elution. The eluents were combined, and the solvent was recovered to obtain a cholesterol concentrate. Cholesterol concentrate was dissolved in a methanol:water:methyl isobutyl ketone (V:V = 9.2:0.8:1) mixture by heating (5 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The mixture was cooled to 0°C and crystallized for 4 hours. After filtration, the solvent was recovered from the filtrate and dried. The resulting filter cake was dissolved in a methanol:water:methyl isobutyl ketone (V:V = 9.2:0.8:1) mixture by heating (1 kg of mixed solvent was added for every 1 kg of cholesterol concentrate). The mixture was cooled to 0°C and crystallized for 4 hours. After filtration, the solvent was recovered from the filtrate and dried. The resulting filter cake was dissolved in a methanol:water (V:V = 90:10) mixture by heating (2 kg of solvent was added for every 1 kg of filter cake). The mixture was cooled to 0°C and recrystallized for 4 hours. After drying under reduced pressure at 50°C for 8 hours, the final product, cholesterol, was obtained. The test results showed that the cholesterol content was 98.5%, β-cholestanol was 0.34%, sterol was 0.12%, encholestanol was 0.22%, 24-cholestipenol was not detected, 3β-hydroxy-5-en-4-methylcholesterol was 0.13%, and total sterol impurities were 0.81%.

[0070] Example of effect 1

[0071] The test results from the examples and comparative examples show that:

[0072] Compared to the examples, Comparative Examples 1-4 used traditional methods to extract cholesterol from fish 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 cholesterol for injection from fish oil. In Comparative Example 5, activated clay was used as an adsorbent, and other conditions were the same as in Example 5, but poor cholesterol separation and enrichment occurred, resulting in a cholesterol purity that could not reach 99% or higher. In Comparative Example 6, the proportion of water and alcohol in the eluent was too high, the eluent polarity was too great, and impurities in the eluent increased, resulting in a cholesterol purity that could not reach 99% or higher. 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 that could not reach 99% or higher, and β-cholesterol exceeded the limit requirements. In Comparative Example 8, the proportion of water in the second crystallization solvent was too high, resulting in a cholesterol purity that could not reach 99% or higher. Only by using Examples 1 to 5 of the present invention can high-purity (over 99%) cholesterol products be successfully obtained from fish oil sources, and the content of other sterols also meets the requirements of the European Pharmacopoeia and the United States Pharmacopoeia.

[0073] 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 from natural fish oil, characterized in that, Includes the following steps: A. Take fish oil, cool it down for winterization, obtain the upper clear liquid and the lower sediment, and perform solid-liquid separation; B. Take the lower sediment and heat it to dissolve it to obtain the winterized fraction; C. Take the winterized fraction and use a chromatography column filled with mixed packing material as the stationary phase for cyclic chromatography; the mixed packing material is selected from an adsorbent and a filter aid that are uniformly mixed in a physical manner; the adsorbent is one or more of alumina, silica gel, and aminosilica gel; the filter aid is one or more of diatomaceous earth, bentonite, and kaolin. D. After the cyclic chromatography is completed, an eluent is added to elute the chromatography column, the eluent is collected, concentrated, and cholesterol concentrate is obtained; the eluent is selected from alkanes, alcohols, and water mixed in a volume ratio of 1:1~5:0.0001~0.0015; in the eluent, the alkanes are one or more of n-hexane, petroleum ether, cyclohexane, dichloromethane, and trichloromethane, and the alcohols are one or more of methanol, ethanol, and isopropanol; E. Take the cholesterol concentrate, add the first crystallization solvent to dissolve it, cool it to crystallize, filter it, discard the filter cake, obtain the filtrate, concentrate the filtrate to recover the solvent to dryness, and obtain the second concentrate. F. Add the first crystallization solvent to the second concentrate to dissolve it, then cool it down to crystallize and obtain crude cholesterol. In steps E and F, the first crystallization solvent is selected from alcohols, ketones and water mixed in a volume ratio of 3~10:1:0.06~0.8; in the first crystallization solvent, the alcohols are one or more of methanol, ethanol and benzyl alcohol, and the ketones are one or more of acetone, butanone and methyl isobutyl ketone. G. Take the crude cholesterol product, add it to the second crystallization solvent to dissolve it, cool it to crystallize it, and dry it to obtain the cholesterol product for injection; the second crystallization solvent is selected from alcohols and their aqueous solutions; the volume fraction of water in the second crystallization solvent is 0~8.0%; in the second crystallization solvent, the alcohol is one or more of methanol and ethanol.

2. The method of claim 1, wherein the natural fish oil source of injectable cholesterol is prepared by, In step D, the mass ratio of the eluent to the mixed packing material is 3~8:1; in step E, the mass ratio of the first crystallization solvent to the cholesterol concentrate is 4~7:1; in step F, the mass ratio of the first crystallization solvent to the cholesterol concentrate is 1~3:1; in step F, the mass ratio of the second crystallization solvent to the crude cholesterol product is 1~3:

1.

3. The method of claim 1, wherein the natural fish oil source of injectable cholesterol is prepared by, In step A, the temperature for the winterization cooling is -10 to 0°C; in step B, the temperature for the lower sediment to be heated and dissolved is 20 to 50°C to obtain the winterization fraction; in step C, the mass ratio of the adsorbent to the winterization fraction is 0.05 to 0.3:1; the mass ratio of the filter aid to the winterization fraction is 0.1 to 0.5:

1.

4. The method of claim 1, wherein the natural fish oil source of injectable cholesterol is prepared by, In step C, the specific conditions for the cyclic chromatography include: temperature 30~50℃, pressure 0.01~1.0 MPa, contact time 2~10h; the winterization fraction is circulated into the chromatography column 1~5 times.

5. The method for preparing injectable cholesterol from natural fish oil according to claim 1, characterized in that, In step A, the solid-liquid separation method comprises filtration; in steps E and F, the temperature for temperature reduction and crystallization is -10 to 25 DEG C; in step G, the temperature for temperature reduction and crystallization is -10 to 25 DEG C; in step G, the drying comprises reduced-pressure drying, and the temperature for the reduced-pressure drying is 50 to 70 DEG C.