Method for extracting lanosterol crude product from wool fat

Abstract

The invention discloses a method for extracting a lanosterol crude product from wool fat. The method comprises the following steps: (1) mixing a first organic solvent with alkali and wool fat, and carrying out saponification reaction to obtain a saponified product; wherein the first organic solvent comprises an aromatic hydrocarbon organic solvent and an alcohol solvent; and (2) carrying out complexation reaction and crystallization on the saponified product obtained in the step (1) to obtain a lanosterol crude product. The extraction method disclosed by the invention is simple in process and low in cost, the obtained lanosterol is high in yield and purity, and the extraction method disclosed by the invention is large in sample separation amount, stable in process and suitable for industrial production.

Description

[0001] This application claims priority to Chinese patent application No. CN2023106723094 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 extracting and separating crude lanosterol from lanolin. Background Art

[0003] Lanolin is mainly composed of esters of alcohols such as sterols, fatty alcohols and triterpenoid alcohols (mainly lanosterol) and higher fatty acids, accounting for about 95% of lanolin. Lanosterol, also known as isocholesterol, has a chemical name of (3β)-8,24-lanostadiene-3-ol, is a white crystal, and belongs to the tetracyclic triterpenoid compound. 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).

[0004] 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 triterpenoid compounds with biological activities such as anti-inflammatory, anti-cancer, and hypolipidemic. Therefore, the research on the extraction and separation of lanosterol from lanolin has a very important position and role. At present, the extraction and separation methods of lanosterol include bromination, extraction, molecular distillation, and preparative chromatography. 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 products of lanosterol and dihydrolanosterol from lanolin by solvent crystallization, without further separation and purification, and the product purity is poor; although the preparative chromatography method (CN200910308433.2, a method for separating and purifying crude lanosterol) can quickly obtain high-purity lanosterol, the processing capacity is small, the equipment cost is high, it is limited to laboratory research, and it is 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 extracting crude lanosterol from lanolin. The yield of the crude lanosterol obtained by the method can reach 6% (calculated based on lanolin) and the purity can reach 65%, which provides possibility for further development of drugs. In addition, the present invention has a large amount of separated samples and a stable process, and is suitable for industrial production.

[0006] The present invention provides a method for extracting crude lanosterol from lanolin, the method comprising the following steps:

[0007] (1) mixing a first organic solvent with an alkali and lanolin to perform a saponification reaction to obtain a saponification product; wherein the first organic solvent comprises an aromatic hydrocarbon organic solvent and an alcohol solvent;

[0008] (2) The saponification product obtained in step (1) is subjected to complexation reaction and crystallization to obtain crude lanosterol.

[0009] According to some embodiments of the present invention, the aromatic organic solvent in step (1) is selected from one or more of benzene, toluene and xylene, preferably one or two of toluene and xylene.

[0010] According to some embodiments of the present invention, the alcohol solvent is selected from one or more of methanol, ethanol, isopropanol, and n-propanol, preferably one or two of methanol and ethanol.

[0011] According to some embodiments of the present invention, the base is selected from alkali metal hydroxides or alkali metal carbonates, preferably potassium hydroxide or sodium hydroxide.

[0012] According to some embodiments of the present invention, in step (1), the volume mass ratio of the first organic solvent to lanolin is (3-5):1 (mL / g), for example, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1.

[0013] According to some embodiments of the present invention, the mass ratio of the alkali to lanolin is (0.15-0.2):1, for example 0.16:1, 0.17:1, 0.18:1, 0.19:1 or 0.2:1.

[0014] According to some embodiments of the present invention, the temperature of the saponification reaction is 60-80°C, for example 65°C, 70°C, 75°C or 78°C.

[0015] According to some embodiments of the present invention, the saponification reaction time is 6 to 8 hours.

[0016] According to some embodiments of the present invention, in step (1), in the first organic solvent, the volume ratio of the aromatic organic solvent to the alcohol solvent is (2.5-5):1, for example, 2.8:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1.

[0017] According to a preferred embodiment of the present invention, in step (1), the first organic solvent comprises toluene and ethanol, preferably, the volume ratio of toluene to ethanol is (2.5-5):1, for example, 2.8:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1.

[0018] According to some embodiments of the present invention, in step (1), after the saponification reaction is completed, a reaction solution is obtained, the reaction solution is cooled to room temperature, and a saponification product is obtained after extraction, separation and decolorization.

[0019] Preferably, the extraction separation and decolorization include: mixing the reaction liquid after the saponification reaction with water and a first extractant to obtain an organic layer and an aqueous layer, extracting the aqueous layer with a second extractant, and combining the organic layers; mixing the organic layer with a sodium hypochlorite solution, decolorizing, washing with saturated brine, separating the liquids to obtain an organic layer, and concentrating the organic layer under reduced pressure to obtain the saponification product.

[0020] In some embodiments, the extraction separation comprises the following steps: mixing the reaction solution with water and a first extractant, stirring and heating to 50-60°C, standing to separate the liquids to obtain an organic layer and an aqueous layer, extracting the aqueous layer with a second extractant at 40-50°C for 1-3 times, and combining the organic layers.

[0021] In some embodiments, the first extractant is selected from aromatic organic solvents and alcohol solvents, preferably one or more of methanol, ethanol, isopropanol, n-propanol, toluene, benzene, and xylene. In some preferred embodiments, the first extractant is a mixture of aromatic organic solvents and alcohol solvents. In some more preferred embodiments, the first extractant is a mixture of ethanol and toluene. In some more preferred embodiments, the volume ratio of ethanol to toluene is 1:(3-5), for example 1:3, 1:3.5, 1:4, 1:4.5 or 1:5.

[0022] In some embodiments, the second extractant is selected from aromatic organic solvents, preferably one or more of benzene, toluene, and xylene.

[0023] In some embodiments, the volume mass ratio of water to lanolin is (3-5):1 (mL / g), for example, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1.

[0024] In some embodiments, the volume mass ratio of the first extractant to lanolin is (4-6):1 (mL / g), for example, 4.5:1, 5:1, 5.5:1 or 6:1.

[0025] In some embodiments, the volume mass ratio of the second extractant to lanolin is (1-3):1 (mL / g), for example, 1:1, 1.5:1, 2:1, 2.5:1 or 3:1.

[0026] In some embodiments, the decolorization comprises the following steps: adding sodium hypochlorite solution to the organic layer obtained by separation to react and decolorize, then adding saturated brine to wash, separating the liquids to obtain an organic layer, and concentrating the organic layer under reduced pressure to obtain a saponification product.

[0027] In some embodiments, the mass concentration of the sodium hypochlorite solution is 8-12%. Preferably, the volume mass ratio of the sodium hypochlorite solution to lanolin is (0.5-2):1 (mL / g), such as 0.8:1, 1:1, 1.5:1, 1.8:1 or 2:1.

[0028] In some embodiments, the volume mass ratio of the saturated saline to lanolin is (0.5-1.5):1 (mL / g), for example, 0.8:1, 1:1, 1.2:1 or 1.5:1.

[0029] According to some embodiments of the present invention, step (1) comprises: adding alkali and lanolin to a first organic solvent, heating to 60-80° C. to carry out a saponification reaction, cooling to room temperature after the reaction is completed, and obtaining a saponification product after separation and decolorization of the reaction solution.

[0030] According to some embodiments of the present invention, the complexing reaction in step (2) comprises: subjecting the saponification product to a complexing reaction with a metal salt in a second organic solvent.

[0031] In some embodiments, the metal salt is selected from metal chlorides, preferably one or more of calcium chloride, magnesium chloride, and zinc chloride, more preferably calcium chloride.

[0032] In some embodiments, the second organic solvent includes an aprotic solvent and a protic solvent. In some embodiments, the aprotic solvent is selected from one or more of ethyl acetate, petroleum ether, n-hexane, and heptane. In some preferred embodiments, the aprotic solvent is ethyl acetate. In some embodiments, the protic solvent is selected from one or both of methanol and ethanol. In some preferred embodiments, the protic solvent is methanol. In some embodiments, the mass ratio of the aprotic solvent to the metal salt is (7 to 9): 1, such as 7.5: 1, 7.8: 1, 8: 1, 8.5: 1 or 8.8: 1. In some embodiments, the mass ratio of the metal salt to the protic solvent is (0.8 to 2): 1, such as 1: 1, 1.2: 1, 1.5: 1, 1.8: 1 or 2: 1.

[0033] According to some embodiments of the present invention, the temperature of the complexation reaction in step (2) is 45°C to 55°C.

[0034] According to some embodiments of the present invention, the time of the complexation reaction in step (2) is 0.5 hour to 1 hour.

[0035] According to some embodiments of the present invention, the solvent used for the crystallization in step (2) is selected from aromatic organic solvents and alcohol solvents. Preferably, the solvent used for the crystallization is a mixture of aromatic organic solvents and alcohol solvents. Preferably, the volume ratio of the aromatic organic solvent to the alcohol solvent is 1: (3 to 6), such as 1: 3, 1: 3.5, 1: 4, 1: 4.5, 1: 5 or 1: 5.5. In some embodiments, the aromatic organic solvent is selected from one or more of benzene, toluene and xylene. In some embodiments, the alcohol solvent is selected from one or more of methanol, ethanol, isopropanol and n-propanol. In some preferred embodiments, the solvent used for the crystallization in step (2) is a mixture of toluene and methanol. Preferably, the volume ratio of toluene and methanol is 1: (3 to 6), such as 1: 3, 1: 3.5, 1: 4, 1: 4.5, 1: 5 or 1: 5.5.

[0036] According to some embodiments of the present invention, in step (2), the mass ratio of the solvent used for the crystallization to lanolin is (0.2-0.6):1, for example, 0.3:1, 0.35:1, 0.4:1, 0.45:1, 0.5:1 or 0.55:1.

[0037] According to some embodiments of the present invention, in step (2), the crystallization temperature is 60-65°C.

[0038] According to some embodiments of the present invention, step (2) comprises the following steps:

[0039] The saponified product obtained in step (1) is added to a second organic solvent, a metal chloride is added, and the mixture is heated to 45° C. to 55° C. for reaction. After the reaction is completed, the mixture is diluted, cooled, filtered, washed, the organic phases are combined and concentrated to obtain an oily substance;

[0040] The oily substance is mixed with a solvent, maintained at 60° C. to 65° C. for 6 to 12 hours, slowly cooled to room temperature, crystallized, filtered, and the filter cake is washed, pulped and filtered to obtain a crude lanosterol product.

[0041] In some embodiments, the beating is to add the filter cake into methanol with a weight of 0.1 to 0.6 times that of the wool fat, and beating at a temperature of 50° C. to 60° C. for 1 to 2 hours.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The raw materials, solvents, extractants and the like used in the method provided by the invention are cheap and readily available, the process is simple, the obtained crude lanosterol has high purity and high yield, and the method of the invention can separate a large amount of samples, has a stable process, and is suitable for industrial production. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below by means of specific implementation methods. It should be understood by those skilled in the art that the specific implementation methods are only used to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0045] Example 1

[0046] This embodiment provides a method for extracting crude lanosterol from lanolin, comprising the following steps:

[0047] 1) Saponification reaction: add 50g lanolin, 150mL toluene and 50mL ethanol solution containing 7.5g potassium hydroxide into a reaction vessel, stir evenly, heat to 80°C for saponification for 6 hours, and cool to room temperature;

[0048] Add 200 mL of water, 200 mL of toluene and 50 mL of ethanol to the reaction solution obtained above, stir evenly, heat to 50°C, stand and separate to obtain an organic layer and an aqueous layer, extract the aqueous layer twice with 100 mL of toluene at 50°C, and combine the organic layers;

[0049] Add 25 mL of 10% sodium hypochlorite aqueous solution to the organic layer obtained above to react and decolorize, then add 75 mL of saturated brine for washing, and concentrate the organic layer obtained by separation under reduced pressure to obtain a saponification product;

[0050] 2) Complexation reaction: add 20g of ethyl acetate to the product obtained in step 1), stir evenly, then add 2.5g of calcium chloride powder and 2.5g of methanol, heat to 40°C and stir to react for 1 hour, then add appropriate amount of ethyl acetate to dilute, cool to room temperature, filter, wash the filter cake with appropriate amount of ethyl acetate, combine the washing liquid and the filtrate and concentrate under reduced pressure to obtain an oily substance;

[0051] 3) Crystallization: Add 20 g of a mixed solvent of toluene and methanol (the volume ratio of toluene and methanol is 1:4) to the oil obtained in step 2), heat to 65 ° C, keep warm for 12 hours, slowly cool to room temperature and stir for 8 hours to precipitate a white solid, filter, add 5 g of methanol to the filter cake, heat to 60 ° C and beat for 2 hours, cool to room temperature, and filter to obtain a white solid powder, thereby obtaining 3.1 g of crude lanosterol.

[0052] Example 2

[0053] This embodiment provides a method for extracting crude lanosterol from lanolin, comprising the following steps:

[0054] 1) Saponification reaction: add 50g lanolin, 200mL xylene and 50mL ethanol solution containing 10g potassium hydroxide into a reaction vessel, stir evenly, heat to 60°C for saponification for 8 hours, and cool to room temperature;

[0055] Add 250 mL of water and 300 mL of xylene to the reaction solution obtained above, stir evenly, heat to 50°C, stand and separate to obtain an organic layer and an aqueous layer, extract the aqueous layer twice with 150 mL of xylene at 50°C, and combine the organic layers;

[0056] Add 50 mL of 12% sodium hypochlorite aqueous solution to the organic layer obtained above to react and decolorize, then add 50 mL of saturated brine for washing, and concentrate the organic layer obtained by separation under reduced pressure to obtain a saponification product;

[0057] 2) Complexation reaction: add 20g of ethyl acetate to the product obtained in step 1), stir evenly, then add 2.5g of calcium chloride powder and 2.5g of methanol, heat to 40°C and stir to react for 1 hour, then add appropriate amount of ethyl acetate to dilute, cool to room temperature, filter, wash the filter cake with appropriate amount of ethyl acetate, combine the washing liquid and the filtrate and concentrate under reduced pressure to obtain an oily substance;

[0058] 3) Crystallization: Add 30 g of a mixed solvent of methanol and xylene (the volume ratio of xylene to methanol is 1:6) to the oil obtained in step 2), heat to 65 ° C, keep warm for 12 hours, slowly cool to room temperature and stir for 8 hours to precipitate a white solid, filter, add 5 g of methanol to the filter cake, heat to 60 ° C and beat for 2 hours, cool to room temperature, and filter to obtain a white solid powder, thereby obtaining 2.95 g of crude lanosterol.

[0059] Example 3

[0060] Compared with Example 1, in step 2), 2.5 g of zinc chloride was used instead of 2.5 g of calcium chloride in the complexation reaction, and the other operation steps were the same as those in Example 1, and finally 2.0 g of crude lanosterol was obtained.

[0061] Example 4

[0062] Compared with Example 1, in step 1) of the saponification reaction, the volume of toluene is 100 mL, the volume of the ethanol solution containing 7.5 g of potassium hydroxide is also 100 mL, and the other operating steps are the same as those in Example 1, and finally 2.05 g of crude lanosterol is obtained.

[0063] Example 5

[0064] Compared with Example 1, in step 1) of the saponification reaction, the volume of toluene was 175 mL, the volume of the ethanol solution containing 7.5 g of potassium hydroxide was 25 mL, and the other operating steps were the same as those in Example 1, and finally 2.4 g of crude lanosterol was obtained.

[0065] Comparative Example 1

[0066] Compared with Example 1, in step 1), 150 mL of ethanol was used instead of 150 mL of toluene in the saponification reaction, and the other operation steps were the same as those in Example 1, and finally 1.5 g of crude lanosterol was obtained.

[0067] In order to further illustrate the beneficial technical effects of each embodiment, the yield of crude lanosterol (calculated as lanolin) prepared in Examples 1 to 5 and Comparative Example 1 and the purity of lanosterol in the crude lanosterol were calculated and tested. The results are shown in Table 1.

[0068] Table 1

[0069] Example Lanosterol crude product yield (%) Lanosterol purity (%) Example 1 6.2％ 65％ Example 2 5.9％ 62％ Example 3 4.0％ 60％ Example 4 4.1％ 56％ Example 5 4.8％ 60％ Comparative Example 1 3.0％ 50％

[0070] It can be seen from the test results in the above table that the yield of the crude lanosterol and the purity of the lanosterol obtained in Examples 1 to 5 are significantly better than those in Comparative Example 1.

[0071] It can be seen that the method of the present application can obtain crude lanosterol with high yield and purity, which can provide favorable conditions for subsequent separation and purification.

[0072] 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 extracting crude lanosterol from lanolin, comprising the following steps: (1) mixing a first organic solvent with an alkali and lanolin to perform a saponification reaction to obtain a saponification product; wherein the first organic solvent comprises an aromatic hydrocarbon organic solvent and an alcohol solvent; and (2) The saponification product obtained in step (1) is subjected to complexation reaction and crystallization to obtain crude lanosterol.

2. The method according to claim 1, characterized in that In step (1), the aromatic hydrocarbon organic solvent is selected from one or more of benzene, toluene and xylene, preferably one or two of toluene and xylene; and / or the alcohol solvent is selected from one or more of methanol, ethanol, isopropanol and n-propanol, preferably one or two of methanol and ethanol; and / or the base is selected from alkali metal hydroxides or alkali metal carbonates, preferably potassium hydroxide or sodium hydroxide.

3. The method according to claim 1 or 2, characterized in that: In step (1), the volume mass ratio of the first organic solvent to lanolin is (3-5): 1 mL / g; and / or the mass ratio of the alkali to lanolin is (0.15-0.2):1; And / or the temperature of the saponification reaction is 60° C. to 80° C., and the time of the saponification reaction is 6 hours to 8 hours.

4. The method according to any one of claims 1 to 3, characterized in that: In step (1), in the first organic solvent, the volume ratio of the aromatic hydrocarbon organic solvent to the alcohol solvent is (2.5-5):

1.

5. The method according to any one of claims 1 to 4, characterized in that: In step (1), the reaction solution after the saponification reaction is extracted and decolorized to obtain a saponification product; preferably, the extraction and decolorization include: The reaction solution after the saponification reaction is mixed with water and the first extractant to obtain an organic layer and an aqueous layer, the aqueous layer is extracted with the second extractant, and the organic layers are combined; the organic layer is mixed with a sodium hypochlorite solution, decolorized, washed with saturated brine, separated to obtain an organic layer, and the organic layer is concentrated under reduced pressure to obtain the saponification product; Preferably, the first extractant is selected from aromatic organic solvents and alcohol solvents, preferably one or more of methanol, ethanol, isopropanol, n-propanol, toluene, benzene, and xylene; and the second extractant is selected from aromatic organic solvents, preferably one or more of benzene, toluene, and xylene.

6. The method according to claim 5, characterized in that The volume mass ratio of the first extractant to lanolin is (4-6): 1 mL / g; and / or the volume mass ratio of the second extractant to lanolin is (1-3): 1 mL / g; and / or the mass concentration of the sodium hypochlorite solution is 8-12%, preferably, the volume mass ratio of the sodium hypochlorite solution to lanolin is (0.5-2): 1 mL / g.

7. The method according to any one of claims 1 to 6, characterized in that: The complexing reaction in step (2) comprises: allowing the saponification product to undergo a complexing reaction with a metal salt in a second organic solvent; Preferably, the metal salt is selected from metal chlorides, preferably one or more of calcium chloride, magnesium chloride, and zinc chloride, more preferably calcium chloride.

8. The method according to claim 7, characterized in that The temperature of the complex reaction is 45 to 55°C, and the time of the complex reaction is 0.5 to 1 hour; and / or the second organic solvent comprises an aprotic solvent and a protic solvent, the aprotic solvent is selected from one or more of ethyl acetate, petroleum ether, n-hexane, and heptane, and the protic solvent is selected from one or both of methanol and ethanol; Preferably, the mass ratio of the aprotic solvent to the metal salt is (7-9):1; the mass ratio of the metal salt to the protic solvent is (0.8-2):

1.

9. The method according to any one of claims 1 to 8, characterized in that: The solvent used for the crystallization in step (2) is selected from an aromatic organic solvent and an alcohol solvent. Preferably, the solvent is a mixture of an aromatic organic solvent and an alcohol solvent. and / or the mass ratio of the solvent used for the crystallization to lanolin is (0.2-0.6):1; And / or the crystallization temperature is 60-65°C.

10. The method according to claim 9, characterized in that The aromatic organic solvent is selected from one or more of benzene, toluene, and xylene, and the alcohol solvent is selected from one or more of methanol, ethanol, isopropanol, and n-propanol; preferably, the volume ratio of the aromatic organic solvent to the alcohol solvent is 1:(3-6).