CN108558983A - A kind of method that separation prepares four kinds of active materials of cycloartenyl ferulate - Google Patents

Abstract

The invention relates to a method for separating and preparing high-purity monomer cycloartenol ferulate, 24-methylenecycloartenol ferulate and campesterol arabic acid from rice bran extract by countercurrent chromatography. The method of ferulic acid ester and sitosterol ferulic acid ester; it adopts countercurrent chromatography to separate and prepare four kinds of active substances such as high-purity cycloartenol ferulic acid ester from rice bran extract, and the solvent system consists of three groups Composition: Petroleum ether or normal alkanes, dichloromethane and acetonitrile or fatty alcohol; volume ratio: 10:0-3:7-12; suitable for the separation and preparation of monomer cycloarpinene by various types of countercurrent chromatography Alcohol ferulate, 24‑methylenecycloartenol ferulate, campesterol ferulate and sitosterol ferulate, can be directly fed into a large number of crude products or synthetic mixtures, separation of cycloartenol The purity of ferulic acid ester and sitosterol ferulic acid ester is over 95%, and the purity of 24-methylenecycloartenyl ferulic acid ester and campesterol ferulic acid ester is over 90%.

Description

A method for separating and preparing four active substances of cycloartenol ferulate

technical field

The invention relates to a high-purity monomer cycloartenol ferulate, 24-methylenecycloartenol ferulate and campesterol arabinate separated from rice bran extract by high-speed countercurrent chromatography. Preparation and separation method of ferulic acid ester and sitosterol ferulic acid ester.

Background technique

Rice is the second largest crop in the world, with an annual output of about 583 million tons. my country is the largest rice producer in the world, with an annual output of more than 200 million tons of rice, and an annual output of rice bran of about 14 million tons. Rice bran is one of the valuable by-products of the rice processing industry. It has been confirmed that a large amount of bioactive components such as sterol ferulate (about 2510-6860 μg/g) are distributed in rice bran and other grain processing wastes, mainly cyclo Carpine alcohol ferulate and campesterol ferulate, of which cycloartenol ferulate accounts for 32%, 24-methylenecycloartenol ferulate accounts for 40%, campesterol Ferulate accounts for 9%, and sitosterol ferulate accounts for 19%. Sterol ferulic acid ester is a derivative formed by dehydrating the hydroxyl group on the C3 position of sterol and ferulic acid. It has better lipophilicity and is easier to pass through biomembranes. The lipid-lowering level, anti-oxidation, anti-inflammation, anti-virus and anti-tumor and other comprehensive biological effects. Therefore, the separation and purification of cycloartenol ferulate and other substances in rice bran is of great significance for the development and utilization of high added value of agricultural and sideline products.

It is reported in existing literature that sterol ferulic acid ester is separated from rice bran by column chromatography, but the monomer with extremely high purity cannot be obtained, while the countercurrent chromatography separation preparation method can obtain the monomer with high purity. Since High-speed Countercurrent Chromatography (High-speed Countercurrent Chromatography, HSCCC) is a continuous high-efficiency and rapid liquid-liquid partition chromatography separation technology developed in recent years without any solid support, it avoids the need for solid supports or carrier bands. Various problems that come - samples are easy to be adsorbed, lost and denatured, HSCCC guarantees higher peak resolution, large separation volume, no sample loss, high recovery rate, mild separation environment, and solvent saving. Countercurrent chromatography can directly enter a large number of crude extraction samples or synthetic mixtures, and the separation results can reach a very high purity. It can even be directly connected to mass spectrometers and other instruments. It has been widely used in the preparation and separation of chemical substances in the fields of biology, medicine, and environmental protection. purification. And cycloartenol ferulate, 24-methylenecycloartenol ferulate, campesterol ferulate and sitosterol ferulate four substances are non-polar compounds, the material structure is extremely Similarly, it is difficult to separate and purify to obtain high-purity monomers.

Contents of the invention

The object of the present invention is to use the rice bran extract extracted with n-hexane as raw material to extract cycloarphanol ferulic acid ester, 24-methylenecycloartenyl ferulic acid ester and campesterol ferulic acid Ester and sitosterol ferulic acid ester are obtained cycloartenyl ferulic acid ester and sitosteryl ferulic acid ester with a purity of more than 95% by high-speed countercurrent chromatography, and 24-methylene cycloartenyl ferulic acid ester with a purity of more than 90% is obtained Pineapple Ferulate and Campesteryl Ferulate.

The scheme of the present invention is: the rice bran extract separates and prepares high-purity cycloartenol ferulate, 24-methylenecycloartenol ferulate, campesterol ferulate and Sitosterol Ferulate. Choose a two-phase non-aqueous solvent system, which is composed of three components, A component is petroleum ether and normal alkanes, B component is dichloromethane, and C component is acetonitrile and fatty alcohol.

Using a non-aqueous solvent system, the upper phase is the stationary phase, and the lower phase is the mobile phase. Under the premise of ensuring that the volume ratio of the upper and lower phases is less than 1, according to the solubility constant, adjust the three groups of A:B:C without destroying the balance of the system. The volume ratio of the points: 10:0-3:7-12, through a countercurrent separation to obtain cycloartenol ferulate, 24-methylene cycloartenol ferulate, campesterol ferulic acid Esters and Sitosterol Ferulate.

The experimental conditions are suitable for room temperature of 10-25°C. Room temperature has a great influence on the separation efficiency. The optimal separation temperature is 15°C. If the temperature is too high, the solvent system will be poorly retained and the separation requirements will not be met.

First, prepare the above solvent system in a separatory funnel according to the volume ratio, shake well and let stand to separate layers. After equilibrating for a period of time, separate the upper and lower phases. Analytical or semi-preparative high-speed countercurrent chromatograph, equipped with NS-1007 pump, 2mL or 20mL injection valve, polytetrafluoroethylene column, column volume is 40mL, 230 or 240mL, 500mL, 8823A-UV ultraviolet detector, Yokogawa 3057 Portable Recorder. The rice bran extract was dissolved in the mobile phase for use. Before sample injection, fill the entire column with the stationary phase, adjust the speed of the main engine to 500-1800 rpm, and pump the mobile phase into the column at a flow rate of 0.5-3.0mL/min. After the dynamic equilibrium of the entire system is established, the sample injection The valve injects the sample; then according to the UV spectrum of the detector, the target component is received.

The purity of the cycloartenol ferulate and sitosterol ferulate extracted by this method can reach more than 95%, and the 24-methylene cycloartenol ferulate and campesterol ferulate The purity reaches more than 90%. It is suitable for the separation and preparation of cycloartenol ferulate, 24-methylenecycloartenol ferulate, campesterol ferulate and sitosterol ferulate by using various types of countercurrent chromatography Monomers can be directly fed into a large number of crude extraction samples or synthetic mixtures, and the separation results can achieve high purity.

Detailed ways

Example 1

Select n-hexane-acetonitrile on a semi-preparative countercurrent chromatograph GS10AB to separate and purify rice bran extract. First, the above solvent components are prepared in a separatory funnel according to a volume ratio of 1:1, shake well and then stand to separate layers. After equilibrating for a period of time, separate the upper phase and the lower phase, take the lower phase as the stationary phase, and the upper phase as the mobile phase. A semi-preparative high-speed countercurrent chromatograph is used, equipped with an NS-1007 pump, a 20mL injection valve, a polytetrafluoroethylene column with a column volume of 240mL, an 8823A-UV ultraviolet detector, and a Yokogawa3057 portable recorder. Weigh 200mg of rice bran extract and dissolve in 5mL upper phase and 5mL lower phase mixed solution for use. Before sample injection, fill the entire column with stationary phase, adjust the speed of the main engine to 800rpm, and pump the mobile phase into the column at a flow rate of 1.5mL/min; after the dynamic equilibrium of the entire system is established, inject the sample through the injection valve; then According to the ultraviolet spectrogram of the detector, the target components are received, and the purity of cycloartenol ferulate and sitosterol ferulate is detected by HPLC to reach more than 95%, and the purity of 24-methylene cycloartenol ferulate and The purity of campesterol ferulic acid ester reaches more than 90%.

Example 2

Select n-heptane-dichloromethane-acetonitrile on the semi-preparative countercurrent chromatograph GS10A to separate the rice bran extract. Set layers. After equilibrating for a period of time, separate the upper phase and the lower phase, take the lower phase as the stationary phase, and the upper phase as the mobile phase. A semi-preparative high-speed countercurrent chromatograph is used, equipped with NS-1007 pump, 20mL injection valve, polytetrafluoroethylene column with a column volume of 230mL, 8823A-UV ultraviolet detector, and Yokogawa 3057 portable recorder. Weigh 128.5mg of rice bran extract and dissolve in 5mL upper phase and 5mL lower phase mixed solution for use. Before sample injection, fill the entire column with stationary phase, adjust the speed of the main engine to 800rpm, and pump the mobile phase into the column at a flow rate of 2.0mL/min; after the dynamic equilibrium of the entire system is established, inject the sample through the injection valve; then According to the ultraviolet spectrogram of the detector, the target components are received, and the purity of cycloartenol ferulate and sitosterol ferulate is detected by HPLC to reach more than 95%, and the purity of 24-methylene cycloartenol ferulate and The purity of campesterol ferulic acid ester reaches more than 90%.

Example 3

Select n-pentane-dichloromethane-acetonitrile on the semi-preparative countercurrent chromatograph EMC-500 to separate and purify the rice bran extract. Let it stand for layering. After equilibrating for a period of time, separate the upper phase and the lower phase, take the lower phase as the stationary phase, and the upper phase as the mobile phase. A semi-preparative high-speed countercurrent chromatograph is used, equipped with NS-1007 pump, 20mL injection valve, polytetrafluoroethylene column with a column volume of 500mL, 8823A-UV ultraviolet detector, and Yokogawa 3057 portable recorder. Weigh 400.2mg of rice bran extract and dissolve in 5mL upper phase and 5mL lower phase mixed solution for use. Before sample injection, fill the entire column with stationary phase, adjust the speed of the main engine to 800rpm, and pump the mobile phase into the column at a flow rate of 2.0mL/min; after the dynamic equilibrium of the entire system is established, inject the sample through the injection valve; then According to the ultraviolet spectrogram of the detector, the target components are received, and the purity of cycloartenol ferulate and sitosterol ferulate is detected by HPLC to reach more than 95%, and the purity of 24-methylene cycloartenol ferulate and The purity of campesterol ferulic acid ester reaches more than 90%.

Example 4

Select n-hexane-ethanol on a semi-preparative countercurrent chromatograph GS10AB to separate and purify rice bran extract. First, the above solvent components are prepared in a separatory funnel at a volume ratio of 5:6, shake well and then stand to separate layers. After equilibrating for a period of time, separate the upper phase and the lower phase, take the lower phase as the stationary phase, and the upper phase as the mobile phase. A semi-preparative high-speed countercurrent chromatograph is used, equipped with an NS-1007 pump, a 20mL injection valve, a polytetrafluoroethylene column with a column volume of 240mL, an 8823A-UV ultraviolet detector, and a Yokogawa3057 portable recorder. Weigh 133.6mg of rice bran extract and dissolve in 5mL upper phase and 5mL lower phase mixed solution for use. Before sample injection, fill the entire column with stationary phase, adjust the speed of the main engine to 800rpm, and pump the mobile phase into the column at a flow rate of 2.0mL/min; after the dynamic equilibrium of the entire system is established, inject the sample through the injection valve; then According to the ultraviolet spectrogram of the detector, the target components are received, and the purity of cycloartenol ferulate and sitosterol ferulate is detected by HPLC to reach more than 95%, and the purity of 24-methylene cycloartenol ferulate and The purity of campesterol ferulic acid ester reaches more than 90%.

Example 5

Select n-hexane-methanol on a semi-preparative countercurrent chromatograph GS10AB to separate and purify rice bran extract. First, the above solvent components are prepared in a separatory funnel at a volume ratio of 1:1, shake well and then stand to separate layers. After equilibrating for a period of time, separate the upper phase and the lower phase, take the lower phase as the stationary phase, and the upper phase as the mobile phase. A semi-preparative high-speed countercurrent chromatograph is used, equipped with an NS-1007 pump, a 20mL injection valve, a polytetrafluoroethylene column with a column volume of 240mL, an 8823A-UV ultraviolet detector, and a Yokogawa3057 portable recorder. Weigh 299.3mg of rice bran extract and dissolve in 5mL upper phase and 5mL lower phase mixed solution for use. Before sample injection, fill the entire column with stationary phase, adjust the speed of the main engine to 800rpm, and pump the mobile phase into the column at a flow rate of 2.0mL/min; after the dynamic equilibrium of the entire system is established, inject the sample through the injection valve; then According to the ultraviolet spectrogram of the detector, the target components are received, and the purity of cycloartenol ferulate and sitosterol ferulate is detected by HPLC to reach more than 95%, and the purity of 24-methylene cycloartenol ferulate and The purity of campesterol ferulic acid ester reaches more than 90%.

Example 6

Petroleum ether-n-butanol was selected to separate and purify rice bran extract on a semi-preparative countercurrent chromatograph GS10AB. First, the above solvent components were prepared in a separatory funnel at a volume ratio of 1:1, shaken and then allowed to stand for stratification. After equilibrating for a period of time, separate the upper phase and the lower phase, take the lower phase as the stationary phase, and the upper phase as the mobile phase. A semi-preparative high-speed countercurrent chromatograph is used, equipped with NS-1007 pump, 20mL injection valve, polytetrafluoroethylene column with a column volume of 240mL, 8823A-UV ultraviolet detector, and Yokogawa 3057 portable recorder. Weigh 150.1mg of rice bran extract and dissolve in 5mL upper phase and 5mL lower phase mixed solution for use. Before sample injection, fill the entire column with stationary phase, adjust the speed of the main engine to 800rpm, and pump the mobile phase into the column at a flow rate of 3.0mL/min; after the dynamic equilibrium of the entire system is established, inject the sample through the injection valve; then According to the ultraviolet spectrogram of the detector, the target components are received, and the purity of cycloartenol ferulate and sitosterol ferulate is detected by HPLC to reach more than 95%, and the purity of 24-methylene cycloartenol ferulate and The purity of campesterol ferulic acid ester reaches more than 90%.

Example 7

Choose methyl tert-butyl ether-n-butanol to separate and purify the rice bran extract on the analytical countercurrent chromatograph GS20, first prepare the above solvent components in a separatory funnel according to the volume ratio of 1:1, shake well and let stand layered. After equilibrating for a period of time, separate the upper phase and the lower phase, take the lower phase as the stationary phase, and the upper phase as the mobile phase. An analytical countercurrent chromatograph is used, equipped with a NS-1007 pump, a 2mL injection valve, a polytetrafluoroethylene column with a column volume of 40mL, an 8823A-UV ultraviolet detector, and a Yokogawa3057 portable recorder. Weigh 30.5mg of rice bran extract and dissolve in 1mL upper phase and 1mL lower phase mixed solution for use. Before sample injection, fill the entire column with the stationary phase, adjust the speed of the main engine to 1800rpm, and pump the mobile phase into the column at a flow rate of 0.5mL/min; after the dynamic equilibrium of the entire system is established, inject the sample through the injection valve; then According to the ultraviolet spectrogram of the detector, the target components are received, and the purity of cycloartenol ferulate and sitosterol ferulate is detected by HPLC to reach more than 95%, and the purity of 24-methylene cycloartenol ferulate and The purity of campesterol ferulic acid ester reaches more than 90%.

Claims (1)

1. a kind of method that separation prepares four kinds of active materials of cycloartenyl ferulate, it is characterised in that：It is to use Counter current chromatography separation from rice rice bran extract prepares cycloartenyl ferulate, 24- methylene basic ring jackfruit alkene Alcohol ferulic acid ester, campesterol ferulic acid ester and sitosterol ferulic acid ester, wherein cycloartenyl ferulate and sitosterol Ferulic acid ester purity is 95% or more, 24- methylene cycloartenyl ferulate and campesterol ferulic acid ester purity is 90% or more, dicyandiamide solution is made of three components：By petroleum ether or n-hexane or normal heptane or pentane, dichloromethane and Acetonitrile or methanol or ethyl alcohol or n-butanol are constituted, and volume ratio is followed successively by： 10：0-3：7-12.