CN102633853B - Method for purifying campesterol from mixed plant sterol - Google Patents

Abstract

The invention discloses a method for purifying campesterol from mixed phytosterols. The method utilizes the different solubility and crystallinity of different phytosterol components in specific organic solvents, uses acetone as an organic solvent to carry out multiple recrystallization processes on the phytosterol mixture, and uses chromatography to detect the content of each phytosterol monomer to obtain Determine the purity of campesterol in all stages of crystallization, and finally extract campesterol with higher purity. The invention provides a method for purifying sterol monomers from mixed phytosterols by using only one organic solvent under large-scale production conditions. The invention has important theoretical and practical significance for the extraction of pure natural products including phytosterols, will play an important role in plant fine chemical industry and analytical chemistry, and has broad application prospects.

Description

A method for purifying campesterol from mixed phytosterols

technical field

The invention relates to the field of biochemical industry or fine chemical industry, in particular to a method for purifying campesterol from mixed phytosterols.

Background technique

Mixed phytosterols are a class of steroidal compounds with similar structures and small differences in physical properties. They are mainly derived from wood pulp tall oil and vegetable oil refining deodorization distillates. The main components are β-sitosterol (β-sitosterol), campesterol ( campesterol), stigmasterol (stigmasterol) and brassicasterol (avenasterol) and so on.

Phytosterols have very important physiological functions, which can effectively reduce low-density cholesterol, reduce the incidence of heart disease, anti-cancer and anti-inflammatory effects. The various physiological functions of phytosterols make them widely used in various fields such as medicine, cosmetics, animal growth agents, food, textile, printing and paper processing industries. Products of phytosterols, phytosterol esters and their derivatives have been commercialized. The high efficiency and safety of phytosterols make phytosterol-related products have very broad development prospects.

Due to the further application of phytosterols in the pharmaceutical industry and scientific research in recent years, higher requirements have been put forward for their purity. How to separate mixed phytosterols into single sterol products and improve their yield and purity is a current research hotspot in the separation and purification of phytosterols. Separation and purification of mixed phytosterols can provide relatively cheap raw materials for the production of steroid drugs. The effective separation of mixed phytosterols is an important research topic in the process of realizing the large-scale production of single-component high-purity phytosterol monomers. Due to the extremely similar structure, it is difficult and cumbersome to separate and purify β-sitosterol, campesterol and brassicasterol from mixed phytosterols to obtain higher purity monomers of each sterol. So far, the methods for extracting sterols include solvent crystallization, complexation, molecular distillation, adsorption, and chromatographic separation. The reliability and economy of industrial production should be considered. Based on the above factors, the solvent crystallization method is generally used for the separation of mixed sterol monomers. Directly use the solubility difference of individual sterols in the solvent to carry out multi-stage fractional crystallization. Although the number of crystallization is more, the process is simple, easy to operate, and the purity can also be high.

At present, there are many studies on the separation and purification of β-sitosterol and stigmasterol at home and abroad, and the purity of the corresponding monomers obtained is relatively high, which can meet the research and market application of their related physiological activities. However, there are few studies on campesterol with the same abundant content, and there are even fewer reports about the separation and refining technology of campesterol. Moreover, the purity of campesterol monomers sold on the market can only reach 60%, and the price is expensive, far from Meet the research and commercial production requirements of its related physiological activities.

Contents of the invention

The purpose of the present invention is to overcome the difficulty in realizing the complete separation of campesterol and sitosterol in the existing methods for separating and purifying sterol monomers, and to provide a method that can be used under large-scale production conditions, using only a kind of organic solvent from mixed plant A method for extracting high-purity campesterol from sterols.

The present invention adopts the solvent crystallization method with simple process and easy operation to separate and purify campesterol. The principle is that different phytosterol components have different solubility in specific organic solvents as the temperature changes, and organic solvents with suitable boiling points can be used for mixing phytosterols. campesterol in multistage fractional crystallization separation.

The technical solution adopted by the present invention to solve its technical problems is as follows:

A method for separating and preparing campesterol from mixed phytosterols is carried out according to the following steps:

① Put the mixed phytosterols in an organic solvent, and after the sterols are completely dissolved in a high-temperature water bath, cool down in the water bath until crystallization occurs;

② Keep crystal growth at constant temperature for several hours;

③ Vacuum filtration under reduced pressure to collect the filter cake and filtrate. The filter cake was air-dried naturally, and the filtrate was decompressed to remove the solvent;

④ Adopt gas chromatography or high performance liquid chromatography to detect the content of campesterol monomer in filter cake and filtrate respectively;

⑤ Carry out the next recrystallization according to the same material ratio, repeat the operation steps many times, the direct product purity can reach 75%, the filtrate is rotary evaporated and the solid part is recovered; according to the different content of campesterol, it can be mixed again and according to the same material ratio for the next crystallization.

The content of campesterol in the mixed phytosterols in the step ① shall not be lower than 25%; the organic solvent is acetone, and the mass-to-volume ratio of the mixed phytosterols to the solvent acetone is 1:5-25 (g/ml).

The dissolving temperature in the water bath in the step ① is 50-70°C, and after the sterol mixture is fully dissolved, the temperature is lowered to 10-25°C.

In the step ②, the crystal growth is maintained at a constant temperature for 12-24 hours.

In the step ③, the filtrate is evaporated under reduced pressure in a water bath at 40-60° C. to remove the solvent, and the solid matter is recovered.

In the gas chromatography in the step ④, the gas chromatography column is a phenyl-methyl stationary phase column, and the temperature program is used for detection.

The HPLC column in the step ④ is an analytical C18 or C8 HPLC column, and the mobile phase is one or a combination of acetonitrile, isopropanol, methanol and water.

The step ⑤ repeats the operation step multiple times, which is to carry out the next recrystallization of the filter cake according to the same process, repeat 2 to 10 times, and recover the solvent to obtain white powdered campesterol with a purity of more than 80% .

The step ⑤ repeats the operation steps multiple times or mixes the phytosterols recovered from the filtrate according to the content of campesterol and carries out the next recrystallization according to the same process, repeating 2 to 10 times, and can be used according to the requirements of the oil extraction sterol content Determine the number of times of recrystallization, and finally the yield of campesterol is more than 75%.

The beneficial effects of the present invention are:

1. Using mixed phytosterols as raw materials, only one organic solvent can be used to obtain high-purity campesterol. Compared with other methods, it has the advantages of low cost, simple process, and easy operation.

2. Real-time monitoring of each recrystallization reaction process by high-performance liquid chromatography, so that the reaction is accurately controlled, the purity of the product is guaranteed and the yield is improved, so that the yield is increased to more than 75%, which is an industrial production of campesterol Conditions are provided.

Detailed ways

The present invention will be further described below in combination with specific embodiments.

Example 1:

1. Weigh 20 grams of phytosterol (purity 97%, of which campesterol content is 32.9%), add 400 ml of acetone of analytical grade to the Erlenmeyer flask with the mass volume ratio of phytosterol and reagent at 1:20, and place at 50°C Dissolve in a water bath. After all the phytosterols are dissolved, the water bath cools down to 15°C, and white crystals slowly precipitate.

2. Put it on the platform and keep it at a constant temperature for 12 hours.

3. Heat preservation and vacuum filtration, collect the filter cake and filtrate, and air-dry the filter cake naturally. The filtrate is evaporated under reduced pressure in a water bath at 40°C to recover sterols and remove organic solvents. The content of campesterol in the sterol recovered from the filter cake and the filtrate was detected by gas chromatography, the gas chromatography column was a phenyl-methyl stationary phase column, nitrogen was used as the carrier gas, and the temperature was programmed to 320°C.

4. Carry out the next recrystallization of the filter cake according to the same material ratio, and recrystallize 8 times sequentially to obtain campesterol with a purity of 80%. The filtrate was processed under reduced pressure in a water bath at 50°C to recover sterols and solvents. The recovered phytosterols were recrystallized according to the same material ratio. The recovered phytosterols were crystallized nine times to obtain campesterol with a purity of 78%. The total yield was 76.3%, and the solvent recovery was 93%.

Example 2:

1. Weigh 10 grams of phytosterol (purity 97%, campesterol content 32.9%), add 100 ml of acetone of analytical grade to a conical flask with a mass volume ratio of phytosterol and reagent of 1:10, and place in a 55°C water bath Dissolve, after all the phytosterols are dissolved, the temperature of the water bath is lowered to 10°C, and white crystals slowly precipitate out.

2. Put it on the platform and keep it at a constant temperature for 18 hours.

3. Insulated vacuum filtration, collecting filter cake and filtrate. The filter cake was air-dried naturally, and the filtrate was rotary-evaporated under reduced pressure in a water bath at 50°C to recover sterols and remove organic solvents. High-performance liquid chromatography was used to detect the content of campesterol in the sterols recovered from the filter cake and filtrate respectively. The chromatographic column type adopted was an analytical ODS C18 column, and the mobile phase was acetonitrile/methanol=6:4, and the flow rate was 1 mL/min. The UV detection wavelength is 206 nm.

4. Carry out the next recrystallization of the filter cake according to the same material ratio, and recrystallize 9 times sequentially, and campesterol with a purity of 81.8% can be obtained. The filtrate was distilled under reduced pressure in a 40°C water bath to recover sterols and solvents. The recovered phytosterols were recrystallized according to the same material ratio. The recovered phytosterols were crystallized nine times to obtain campesterol with a purity of 79.5%. The total yield was 75.3%, and the solvent recovery was 95%.

Example 3:

1. Weigh 5 grams of phytosterol (purity 97%, campesterol content 32.9%), add 75 ml of acetone of analytical grade to the Erlenmeyer flask with the mass volume ratio of phytosterol and reagent at 1:15, and place in a 60°C water bath Dissolve, after all the phytosterols are dissolved, the temperature of the water bath is lowered to 20°C, and white crystals slowly precipitate out.

2. Put it on the platform and keep it at a constant temperature for 24 hours.

3. Heat preservation and vacuum filtration, collect the filter cake and filtrate, air-dry the filter cake naturally, and the filtrate is rotatated under reduced pressure in a water bath at 45°C to recover sterols and remove organic solvents. Use high-performance liquid chromatography to detect the content of campesterol in the sterols recovered from the filter cake and filtrate respectively. The chromatographic column type used is an analytical C8 column, and the mobile phase is acetonitrile/isopropanol/water=90:7:3, and the flow rate is 1 mL/min, UV detection wavelength 208 nm.

4. Carry out the next recrystallization of the filter cake according to the same material ratio, and recrystallize 8 times sequentially, and campesterol with a purity of 81.5% can be obtained. The filtrate was processed under reduced pressure in a water bath at 55°C to recover sterols and solvents. The recovered phytosterols were recrystallized according to the same material ratio. The recovered phytosterols were crystallized nine times to obtain campesterol with a purity of 79.6%. The total yield was 75.8%, and the solvent recovery was 95%.

Example 4

1. Weigh 15 grams of phytosterol (purity 97%, campesterol content 32.9%), add 150 ml of acetone of analytical grade to the Erlenmeyer flask with the mass volume ratio of phytosterol and reagent at 1:10, and place in a 70°C water bath Dissolve, after all the phytosterols are dissolved, the temperature of the water bath is lowered to 20°C, and white crystals slowly precipitate out.

2. Put it on the platform and keep it at a constant temperature for 18 hours.

3. Heat preservation and vacuum filtration, collect the filter cake and filtrate, air-dry the filter cake naturally, and the filtrate is rotatated under reduced pressure in a water bath at 45°C to recover sterols and remove organic solvents. High-performance liquid chromatography was used to detect the content of campesterol in the sterols recovered from the filter cake and filtrate respectively. The chromatographic column type used was an analytical C8 column, and the mobile phase was acetonitrile/methanol/water=6:3:1, and the flow rate was 1.5 mL /min, UV detection wavelength 208 nm.

4. The filter cake is subjected to the next recrystallization according to the same material ratio, and recrystallized 10 times sequentially, and campesterol with a purity of 82.3% can be obtained. The filtrate was processed under reduced pressure in a water bath at 50°C to recover sterols and solvents. The recovered phytosterols were recrystallized according to the same material ratio. The recovered phytosterols were crystallized nine times to obtain campesterol with a purity of 78.8%. The total yield was 76.8%, and the solvent recovery was 95%.

Claims (1)

1. from mixed phytosterin, be separated the method preparing campesterol, it is characterized in that carrying out according to following step:

1. mixed phytosterin is placed in organic solvent, high temperature bath is after sterol all dissolves, and water-bath is cooled to crystallization;

2. constant temperature growing the grain a few hours are kept;

3. decompress filter, collects filter cake and filtrate; Filter cake natural air drying, filtrate decompression removes solvent;

4. vapor-phase chromatography or high performance liquid chromatography is adopted to detect the content of campesterol monomer in filter cake and filtrate respectively;

5. carry out recrystallization next time according to identical material ratio, repeatedly repetitive operation step, direct product purity can reach 75%;

Described step 1. in the content of campesterol must not lower than 25% in mixed phytosterin; Organic solvent is acetone, and the quality of mixed phytosterin and solvent acetone and volume ratio are 1:5 ~ 25 grams per milliliter;

Described step 1. middle water-bath solvent temperature is 50 ~ 70 DEG C, after sterol mixture all dissolves, is cooled to 10 ~ 25 DEG C;

Described step is 2. middle keeps constant temperature growing the grain 12-24 hour;

Described step 3. in filtrate reduce pressure in 40 ~ 60 DEG C of water-baths rotary evaporation remove solvent, reclaim solid matter;

Described step 4. middle vapor-phase chromatography adopts gas chromatographic column to be PHENYL-METHYL stationary phase chromatographic column, adopts temperature-programmed mode to detect;

Described step 4. in liquid-phase chromatographic column in high performance liquid chromatography, be analysis mode C18 or C8 performance liquid chromatographic column, moving phase is one or both the combination in acetonitrile, Virahol, first alcohol and water;

Described step 5. repeatedly repetitive operation step be by last step 3. in filter cake according to same technique, repeatedly carry out from step 1. to recrystallization 3., repeat 2 ~ 10 times, filter cake can obtain the campesterol of white powder after removing solvent acetone, purity reaches more than 80%;

Described step 5. repeatedly repetitive operation step, also comprise by step 3. in the solid part that reclaims after rotary evaporation of filtrate utilize step 4. to detect campesterol content after, mix according to campesterol content, and according to same technique, repeatedly carry out from step 1. to 3. recrystallization, repeat 2 ~ 10 times, also purification is carried out to the campesterol in filtrate; Total recovery is more than 75%.