CN103772472B - A kind of method utilizing high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary - Google Patents

Abstract

The invention discloses a method for separating and purifying fritillaria from fritillary by using high-speed countercurrent chromatography, comprising: 1) taking dried fritillaria bulbs to prepare total alkaloids of fritillaria; 2) adopting high-speed countercurrent chromatography to separate and purify fritillaria octane; a mixed solution made of n-hexane, ethyl acetate, methanol, and water in a volume ratio of 2 to 4:5:2 to 3:5 is used as the stationary phase (upper layer) and mobile phase (lower layer); The alkaloid powder is dissolved in the same amount of mobile phase and stationary phase, the sample is injected by the injection valve, the chromatogram is observed, the fraction is collected when the target peak appears, and the solvent is evaporated to obtain fritillaria. The invention does not need to use a solid phase carrier, has no irreversible adsorption, and has a simple, fast and efficient method, and the obtained target compound has high purity.

Description

A method for separating and purifying fritillaria from fritillaria by high-speed countercurrent chromatography

technical field

The invention belongs to the technical field of natural medicinal chemistry, and relates to a method for preparing fritillaria from traditional Chinese medicine fritillaria, in particular to a method for separating and purifying fritillaria from fritillary by using high-speed countercurrent chromatography (HSCCC).

Background technique

The isosteroid alkaloid cyclopamine (cyclopamine) found in Liliaceae Fritillaria, Veratrum and other plants is one of the most concerned anti-tumor natural products in the world. Studies have shown that it is a A Hedgehog pathway inhibitor, which has good inhibitory effects in vivo and in vitro on various cancers such as non-small cell lung cancer, gastric cancer, and pancreatic cancer. Since the content of cyclopamine in plants is low and the total chemical synthesis is very difficult, it is a feasible strategy to isolate cyclopamine analogues from plants and obtain cyclopamine through semi-synthesis. At present, some people have successfully converted peimisine into cyclopamine. Therefore, fritillaria is a compound with great development and utilization value.

Fritillaria is a commonly used traditional Chinese medicine for relieving cough and reducing phlegm. It comes from the bulbs of various Fritillaria species in the genus Fritillaria of the family Liliaceae. The 2010 edition of "Chinese Pharmacopoeia" contains five categories of Fritillaria: Chuan Fritillaria, Zhejiang Fritillaria, Flat Fritillaria, Yi Beimu and Hubei Fritillaria. All kinds of Fritillaria contain a certain amount of isosteroidal alkaloids of steriloid, among which the representative compound is fritillaria, which is distributed in almost all varieties of Fritillaria. Currently, column chromatography is the main method for separating and purifying fritillaria from fritillaria, but the separation cycle is long, the operation is cumbersome, the consumption of organic reagents is large, and the recovery efficiency is low.

High-speed countercurrent chromatography is a separation technique based on the partitioning of samples between immiscible two-phase solvents. This technique does not use solid adsorbents. It is a liquid-liquid partition chromatography technique that uses In a two-phase solvent system, one phase acts as the stationary phase for chromatographic separations, and the other acts as the mobile phase. High-speed countercurrent chromatography does not use a solid carrier, so compared with traditional column chromatography technology, there is no irreversible adsorption of samples and contamination of samples. Compared with traditional column chromatography, it also has a series of advantages such as large preparation amount, short separation process, simple sample pretreatment, solvent saving, economical and effective, and a wide range of solvent system choices. At present, high-speed countercurrent chromatography technology has been widely used in the fields of medicine and chemical industry, especially in the separation and purification of natural products, which has been regarded as a new and effective separation technology. The optimization of suitable solvent system (mobile phase, stationary phase), rotation speed, flow rate, sample volume and other parameters plays a key role in the preparation of high-speed countercurrent chromatography, especially the distribution coefficient K value of each component in the sample determines It depends on whether the solvent system is suitable or not, so the determination of K value is an important part of selecting the solvent system.

Contents of the invention

Purpose of the invention: In view of the deficiencies in the prior art, the purpose of the present invention is to provide a method for separating and purifying fritillaria from fritillaria by high-speed countercurrent chromatography, which has the advantages of short preparation time, simple operation, and less sample loss. , and can obtain fritillary octane monomer with higher purity.

Technical solution: In order to realize the above-mentioned purpose of the invention, the technical solution adopted in the present invention is:

A method for separating and purifying fritillaria from fritillaria by high-speed countercurrent chromatography, comprising the following steps:

1) Take dried fritillary bulbs, crush them into coarse powder, add ammonia water to alkalinize them, extract them under hot reflux with alcohol, combine the extracts, concentrate under reduced pressure to obtain extracts; dissolve the extracts with 0.1M hydrochloric acid, filter to obtain acid solution, and The acid solution is first extracted and degreased with petroleum ether, the acid water layer is adjusted to pH 9~10 with 10% sodium hydroxide, and then extracted several times with dichloromethane, the dichloromethane solution is combined, concentrated and dried under reduced pressure to obtain the total organism of Fritillaria alkali;

2) Use high-speed countercurrent chromatography to separate and purify fritillaria; make a mixed solution of n-hexane, ethyl acetate, methanol, and water in a volume ratio of 2~4:5:2~3:5 and place it in a separatory funnel. 12h; take the upper layer as the stationary phase, and the lower layer as the mobile phase; pump the stationary phase first, wait until the stationary phase is filled with the column of the high-speed countercurrent chromatography, stop pumping the stationary phase, and then pump the mobile phase, when the mobile phase flows out and the baseline is balanced , take the total alkaloid powder of Fritillaria japonicus and dissolve it in the same amount of mobile phase and stationary phase, inject the sample through the injection valve, observe the chromatogram, collect fractions when the target peak appears, and evaporate the solvent to obtain Fritillaria sine.

In step 1), the concentration of alcohol in the extraction method is 70%-95%.

In step 2), the high-speed countercurrent chromatography operation is as follows: the temperature of the constant temperature circulator is set to 35°C, and the set wavelength is 214nm; The flow rate of the phase is 1.5~2mL/min.

Beneficial effects: Compared with the prior art, the method of the present invention for separating and purifying fritillaria from Fritillaria by using high-speed countercurrent chromatography, using high-speed countercurrent chromatography separation technology, has short preparation time, simple operation, less sample loss, and can Obtain fritillin monomer with higher purity. Has very good practicality.

Description of drawings

Fig. 1 is the high-speed countercurrent chromatogram of embodiment 1 gained;

Fig. 2 is the high performance liquid chromatogram of Fritillaria total alkaloids of embodiment 1 gained;

Fig. 3 is the high performance liquid chromatogram of fritillin standard product;

Fig. 4 is the high performance liquid chromatogram of fritillaria in embodiment 1 gained;

Fig. 5 is the secondary mass spectrogram of fritillaria obtained in embodiment 1.

Detailed ways

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

Example 1

A method for separating and purifying fritillaria from fritillaria by high-speed countercurrent chromatography, comprising the following steps:

(1) Preparation of crude extract of total alkaloids from Taibai Fritillaria

Take 100g of Taibai Fritillaria, crush it into a coarse powder, add ammonia water to make sure that the medicinal materials have just been submerged, alkalize for 1 hour, heat reflux with 700mL of 95% ethanol for 4 times, each time for 1.5 hours, combine the extracts from 4 times, and concentrate under reduced pressure Get the extract. Dissolve the extract with 50mL 0.1M hydrochloric acid, filter the acid solution, extract and degrease the acid solution with petroleum ether first, then adjust the pH to 9~10 with 10% sodium hydroxide, and then extract with dichloromethane 4 times, dichloromethane The amount of methane was 100mL, 100mL, 80mL, 60mL in turn, the dichloromethane layers were combined, concentrated and dried under reduced pressure to obtain 691mg of total alkaloid powder of Fritillaria japonicus.

(2) High-speed countercurrent chromatographic separation

The application of high-speed countercurrent chromatography TBE-300B (Shanghai Tongtian Biotechnology Co., Ltd.) separation and purification fritillaria.

Mix n-hexane, ethyl acetate, methanol, and water in a ratio of 4:5:3:5, that is, n-hexane 400mL, ethyl acetate 500mL, methanol 300mL, and water 500mL to make a mixed solution, put it in a separatory funnel, and let it stand for 12 hours spare. Take the upper layer as the stationary phase and the lower layer as the mobile phase. Take 50 mg of the total alkaloid sample of Fritillaria in the above method, dissolve it in 6 mL equal volumes of the upper and lower phases, and ultrasonicate for 40 min to fully dissolve the sample and remove air bubbles. Set the temperature of the constant temperature circulator to 35°C, turn on the ultraviolet detector and N2000 chromatographic workstation, and set the wavelength to 214nm. Start pumping the stationary phase at a flow rate of 2mL/min, then slowly adjust the flow rate to 20mL/min, stop pumping the stationary phase after the stationary phase fills the pipeline, turn on the main engine and adjust the speed to 800rpm, and pump in at a flow rate of 2mL/min Mobile phase, when the mobile phase flows out and the baseline is balanced, inject the sample through the injection valve, record the chromatogram (Figure 1), collect the fractions (10mL/tube) with a fraction collector, and use a high-performance liquid high performance liquid chromatography) to detect each fraction.

(3) Collection and purity determination of target compounds

The HPLC detection conditions are as follows: Chrom-Matrix C18 (250mm × 4.6mm I.D, 5μm), column temperature 40°C; mobile phase is 2mM ammonium bicarbonate aqueous solution (phase A) and 2mM ammonium bicarbonate/40% water/60% acetonitrile ( Phase B). Flow rate 1.0mL/min; dual wavelength (203 and 215nm) detection, column temperature 40°C. The injection volume was 20 μL. Gradient elution steps: 0min, 35%B; 15min, 65%B; 23min, 95%B; 35min, 95%B. Under this chromatographic condition, the total alkaloids of Fritillaria (Figure 2) and the reference substance of Fritillaria (Figure 3) were analyzed, and it was shown that the peak with a retention time of 17.8 min in the total alkaloids of Fritillaria was Fritillaria.

Collection of the target compound: After detecting the fractions collected sequentially by the fraction collector, it was found that the target compound (filicin) elutes between 54 and 75 min of the high-speed countercurrent chromatography (Figure 1).

Purity determination of the target compound: The fractions between 54 and 75 min of high-speed countercurrent chromatography were collected, and the purity measured by high-performance liquid chromatography area normalization method was 96.3% (Figure 4), which was confirmed by the molecular weight and characteristic ions in the secondary mass spectrometry It was identified as fritillaria (Figure 5), and the solvent was evaporated to obtain 5.3 mg of fritillaria.

Example 2

A method for separating and purifying fritillaria from fritillaria by high-speed countercurrent chromatography, comprising the following steps:

(1) Preparation of crude extract of total alkaloids from Fritillaria wabu

Take 100 g of dried and pulverized Fritillaria wabu coarse powder (passed through a 20-mesh sieve), and prepare total alkaloids according to the method in Example 1 to obtain 578 mg of total alkaloid powder.

(2) High-speed countercurrent chromatographic separation

The application of high-speed countercurrent chromatography TBE-300B (Shanghai Tongtian Biotechnology Co., Ltd.) separation and purification fritillaria.

The two-phase solvent system for high-speed countercurrent chromatography is n-hexane-ethyl acetate-methanol-water (3:5:3:5, v/v/v). Take 50 mg of the total alkaloid powder of fritillaria obtained in step 1 and dissolve it in 6 mL of the same amount of upper phase and lower phase, and inject the sample through the injection valve. The separation and purification, purity detection and structure verification methods and steps of fritillaria are the same as in the examples 1. The purity was 97.2%, and the solvent was evaporated to dryness to finally obtain 4.4 mg of fritillaria.

Example 3

Choose a solvent system with a suitable partition coefficient. It is generally believed that the partition coefficient K value between 0.5 and 2 is better for separation.

Determination of K value: Take Fritillaria alkaloids (about 1mg) in a 4mL EP test tube, use a two-phase solvent system that has reached distribution equilibrium in advance, take 0.5mL of the upper phase and lower phase of the same volume to dissolve it, shake vigorously to let It is fully mixed, and after the separation equilibrium is reached, the upper phase and the lower phase sample solutions of the same volume are respectively taken in two test tubes, dried with nitrogen, and 0.5mL of chromatographic methanol is added for dissolution, and the HPLC detection is carried out. The peak areas are respectively recorded as A _up and A _down , the distribution coefficient K is calculated according to the following formula: K=A _up /A _down , the results are shown in Table 1.

It can be seen from Table 1 that in the dichloromethane-methanol-water system, the measured partition coefficient is too small, and it can be seen that the solubility in the stationary phase is too small. During countercurrent chromatographic separation, the target compound is eluted with the mobile phase. It is very short, there is not enough time for separation, and in the actual HSCCC separation, it is also confirmed that the target sample cannot be separated well, and the system of adding hydrochloric acid (aq, 0.01M) has a large distribution coefficient and does not Suitable as an isolated solvent system. The present invention finds that in the n-hexane-ethyl acetate-methanol-water solvent system, the measured distribution coefficient is relatively suitable, so this system is selected as the solvent system for separating fritillary octane. Through experiments, n-hexane-ethyl acetate-methanol-water (2~4:5:2~3:5) can separate and obtain the target compound with higher purity compared with other ratio systems.

Table 1 Distribution coefficients (K) of target components in different solvent systems

Claims (2)

1. utilize a method for high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary, it is characterized in that, comprise the following steps:

(1) Bulbus Fritillariae taipaiensis total alkaloids crude extract preparation

Get Bulbus Fritillariae taipaiensis 100g, be ground into meal, add ammoniacal liquor and be advisable just not have medicinal material, alkalize 1 hour, with 95% ethanol 700mL thermal backflow 4 times, each 1.5 hours, merge No. 4 extracting solutions, concentrating under reduced pressure obtained medicinal extract; Medicinal extract is used 50mL0.1M dissolving with hydrochloric acid, filter to obtain acid solution, acid solution is first used petroleum ether extraction degreasing, then pH is adjusted to be 9 ~ 10 with 10% sodium hydroxide, use dichloromethane extraction again 4 times, quantity of dichloromethane is 100mL, 100mL, 80mL, 60mL successively, combined dichloromethane layer, and concentrating under reduced pressure is dry obtains fritillaria total alkaloids powder 691mg;

(2) high speed adverse current chromatogram is separated

Application high speed adverse current chromatogram TBE-300B separation and purification peimisine;

Normal hexane, ethyl acetate, methyl alcohol, water are made into mixing solutions are placed in separating funnel in the ratio of 4:5:3:5 and normal hexane 400mL, ethyl acetate 500mL, methyl alcohol 300mL, water 500mL, leaves standstill 12h for subsequent use; Get upper strata as stationary phase, lower floor is as moving phase; Get fritillaria total alkaloids sample 50mg in aforesaid method, be dissolved in 6mL equivalent respectively upper and lower mutually in, ultrasonic 40min, abundant sample dissolution and drain bubble; Setting constant temperature circulating actuator temperature is 35 DEG C, opens UV-detector and N2000 chromatographic working station, and to arrange wavelength be 214nm; Start to pump into stationary phase with the flow velocity of 2mL/min, then slowly regulate flow velocity to 20mL/min, to be fixed be full of pipeline mutually after, stop pumping into stationary phase, open main frame and turn speed to 800rpm, moving phase is entered with the flow pump of 2mL/min, flow out when there being moving phase and after baseline balance, by sampling valve sample introduction, record color atlas, collect cut 10mL/ pipe with run tank, detect each cut with Agilent 1260 type high performance liquid chromatograph;

(3) collection of target compound and purity testing

HPLC testing conditions is as follows: Chrom-Matrix C18 250mm × 4.6mm I.D, 5 μm, column temperature 40 DEG C; Moving phase is 2mM ammonium bicarbonate aqueous solution A phase and 2mM bicarbonate of ammonia/40% water/60% acetonitrile B phase; Flow velocity 1.0mL/min; Dual wavelength 203 and 215nm detect, column temperature 40 DEG C; Sample size is 20 μ L; Gradient elution step: 0min, 35%B; 15min, 65%B; 23min, 95%B; 35min, 95%B; Under this chromatographic condition, fritillaria total alkaloids and peimisine reference substance are analyzed, show that retention time in fritillaria total alkaloids is that the peak of 17.8min is peimisine;

The collection of target compound: through detecting the cut that run tank is collected successively, finds that target compound peimisine flows out between 54 ~ 75min of high speed adverse current chromatogram;

The purity testing of target compound: collect the cut between high speed adverse current chromatogram 54 ~ 75min, it is 96.3% that high performance liquid chromatography area normalization method records purity, and confirming it for peimisine through second order ms middle-molecular-weihydroxyethyl and characteristic ionic, solvent evaporated obtains peimisine 5.3mg.

2. utilize a method for high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary, it is characterized in that, comprise the following steps:

(1) watt cloth fritillaria total alkaloids crude extract preparation

Watt cloth bulb of fritillary meal getting drying and crushing good crosses 20 mesh sieve 100g, prepares total alkaloids, obtain total alkaloids powder 578mg by method in claim 1;

(2) high speed adverse current chromatogram is separated

Application high speed adverse current chromatogram TBE-300B separation and purification peimisine;

High speed adverse current chromatogram two phase solvent system is n-hexane-ethyl acetate-methanol-water 3:5:3:5, v/v/v/v; The fritillaria total alkaloids powder 50mg getting step 1 gained is dissolved in the upper and lower phase of 6mL equivalent, and by sampling valve sample introduction, the separation and purification of peimisine, purity detecting and structural identification method, step are with claim 1; Purity is 97.2%, and solvent evaporated finally obtains peimisine 4.4mg.