CN103772472A - Method for separating and purifying peimisine from fritillaria by using high-speed countercurrent chromatography - Google Patents
Method for separating and purifying peimisine from fritillaria by using high-speed countercurrent chromatography Download PDFInfo
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- CN103772472A CN103772472A CN201410005295.1A CN201410005295A CN103772472A CN 103772472 A CN103772472 A CN 103772472A CN 201410005295 A CN201410005295 A CN 201410005295A CN 103772472 A CN103772472 A CN 103772472A
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Abstract
The invention discloses a method for separating and purifying peimisine from fritillaria by using a high-speed countercurrent chromatography. The method comprises the steps: (1) preparing total alkaloid of fritillaria by using dry fritillaria bulbs; (2) separating and purifying peimisine by using high-speed countercurrent chromatography; with a mixed solution as a stationary phase (an upper layer) and a mobile phase (a lower layer), dissolving the total alkaloid powder of the fritillaria into the equivalent mobile phase and stationary phase, introducing a sample through an injection valve, observing a chromatogram, collecting fractions when a target peak appears, and volatizing a solvent to obtain the peimisine, wherein the mixed solution is prepared from n-hexane, ethyl acetate, methyl alcohol and water according to the volume ratio of (2-4):5:(2-3):5. The method is free of solid-phase carriers and irreversible adsorption, simple, rapid, efficient and high in purity of the obtained target compound.
Description
Technical field
The invention belongs to Natural Medicine Chemistry technical field, relate to a kind of method of preparing peimisine from the Chinese medicine bulb of fritillary, particularly one is utilized the method for high-speed countercurrent chromatography (HSCCC) separation and purification peimisine from the bulb of fritillary.
Background technology
The different tetrahydroisoquinoline alkaloid cyclopamine of Jie Li Lu class (cyclopamine) of finding from the plants such as Liliaceae Fritillaria, Veratrum is one of current Antitumor Natural Products of greatest concern in the world, research shows that it is a kind of Hedgehog pathway inhibitor, and kinds cancer is had to good inside and outside restraining effect as nonsmall-cell lung cancer, cancer of the stomach, carcinoma of the pancreas etc.Because cyclopamine content in plant is lower, simultaneously chemistry is complete synthesis very difficult, from plant separating ring bar amine analogue, be more feasible strategy by the semi-synthetic cyclopamine that obtains again.At present existing people successfully changes into cyclopamine by peimisine (peimisine).Therefore, peimisine is a kind of compound that has value of exploiting and utilizing.
The bulb of fritillary is relieving cough and reducing sputum conventional Chinese medicine, derives from the bulb of the multiple bulb of fritillary of Liliaceae (Liliaceae) Fritillaria (Fritillaria).2010 version " Chinese Pharmacopoeia " recorded the five large class bulbs of fritillary: Unibract Fritillary Bulb, Thunberg Fritillary Bulb, Ussuriensis Fritillary Bulb, Sinkiang Fritillary Bulb and Hupeh Fritillary Bulb.All kinds of bulbs of fritillary all contain the different tetrahydroisoquinoline alkaloid of a certain amount of Jie Li Lu class, and wherein representational compound is peimisine, almost in all bulb of fritillary kinds, have distribution.At present, separation and purification peimisine from the bulb of fritillary, existing method mainly contains column chromatography, but its separation cycle is long, complex operation, and organic reagent consumption is large and organic efficiency is low.
High speed adverse current chromatogram is a kind of isolation technique based on sample distributional effects between immiscible two-phase solvent, this technology does not adopt solid adsorbent, it is a kind of liquid luquid partition chromatography technology, utilize the stationary phase as chromatographic separation in immiscible two phase solvent system, another is as moving phase.High speed adverse current chromatogram does not have the use of solid-state carrier, therefore, compared with traditional column chromatography technology, does not have the impact such as the irreversible adsorption of sample and the pollution on sample.The series of advantages such as it also has that preparation amount is large, sepn process is short, sample pretreatment is simple, saves solvent compared with traditional column chromatography, economical and effective, solvent system selection scope are large.High-speed countercurrent chromatography has been widely used in the fields such as medication chemistry at present, especially in the isolation and purification process of natural product, has been considered to be a kind of novel effective isolation technique.And suitable solvent systems (moving phase, stationary phase), rotating speed, flow velocity, the isoparametric optimization of sample size play critical effect in high speed adverse current chromatogram preparation, particularly in sample, the partition ratio K value of each component is determining that whether suitable solvent systems is, and therefore the mensuration of K value is the important step of selective solvent system.
Summary of the invention
Goal of the invention: for the deficiencies in the prior art, the object of this invention is to provide a kind of method of utilizing high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary, possesses preparation time short, simple to operate, the advantages such as sample loss is few, and can obtain the peimisine monomer that purity is higher.
Technical scheme: in order to realize foregoing invention object, the technical solution used in the present invention is:
A method of utilizing high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary, comprises the following steps:
1) get dry bulb of fritillary bulb, be ground into meal, add liquid ammonia alkalinization, with alcohol thermal backflow extraction, united extraction liquid, concentrating under reduced pressure obtains medicinal extract; By medicinal extract 0.1M dissolving with hydrochloric acid, filter to obtain acid solution, acid solution is first used to petroleum ether extraction degreasing, get sour water layer 10% sodium hydroxide and adjust pH to 9 ~ 10, then with dichloromethane extraction for several times, combined dichloromethane liquid, the dry fritillaria total alkaloids that to obtain of concentrating under reduced pressure;
2) adopt high speed adverse current chromatogram separation and purification peimisine; By normal hexane, ethyl acetate, methyl alcohol, water be by volume 2 ~ 4:5:2 ~ 3:5 be made into mixing solutions be placed in separating funnel leave standstill 12h; Get upper strata as stationary phase, lower floor is as moving phase; First pump into stationary phase, treat that stationary phase is full of high-speed counter-current chromatograph pillar, stop pumping into stationary phase, then pump into moving phase, when have moving phase to flow out and baseline balance after; get fritillaria total alkaloids powder and be dissolved in moving phase and the stationary phase of equivalent; by sampling valve sample introduction, observe color atlas, in the time that target peak occurs, collect cut; volatilize solvent, obtain peimisine.
In step 1), in described extracting method, the concentration of alcohol is 70%～95%.
Step 2) in, high speed adverse current chromatogram is operating as: the constant temperature circulating actuator temperature of setting is 35 ℃, and the wavelength of setting is 214nm; The flow velocity that pumps into stationary phase is 5 ~ 20mL/min, and engine speed is 800 ~ 850rpm, and the flow velocity of pump moving phase is 1.5 ~ 2mL/min.
Beneficial effect: compared with prior art, the method for utilizing high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary of the present invention, utilizes high speed adverse current chromatogram isolation technique, preparation time is short, simple to operate, sample loss is few, and can obtain the peimisine monomer that purity is higher.There is good practicality.
Accompanying drawing explanation
Fig. 1 is the high speed adverse current chromatogram figure of embodiment 1 gained;
Fig. 2 is the fritillaria total alkaloids high-efficient liquid phase chromatogram of embodiment 1 gained;
Fig. 3 is the high-efficient liquid phase chromatogram of peimisine standard substance;
Fig. 4 is the peimisine high-efficient liquid phase chromatogram of embodiment 1 gained;
Fig. 5 is the peimisine second order ms figure of embodiment 1 gained.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further.
Embodiment 1
A method of utilizing high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary, comprises the following steps:
(1) Bulbus Fritillariae taipaiensis total alkaloids crude extract preparation
Get Bulbus Fritillariae taipaiensis 100g, be ground into meal, add ammoniacal liquor just not have medicinal material to be advisable, alkalize 1 hour, with 95% ethanol 700mL thermal backflow 4 times, each 1.5 hours, merge No. 4 times extracting solution, concentrating under reduced pressure obtains medicinal extract.By 50mL 0.1M dissolving with hydrochloric acid for medicinal extract, filter to obtain acid solution, acid solution is first used to petroleum ether extraction degreasing, then adjusting pH with 10% sodium hydroxide is 9 ~ 10, use again dichloromethane extraction 4 times, quantity of dichloromethane is 100mL, 100mL, 80mL, 60mL successively, combined dichloromethane layer, and concentrating under reduced pressure is dried to obtain fritillaria total alkaloids powder 691mg.
(2) high speed adverse current chromatogram separates
Application high speed adverse current chromatogram TBE-300B(Shanghai Tongtian Biotechnology Co., Ltd.) separation and purification peimisine.
Be that normal hexane 400mL, ethyl acetate 500mL, methyl alcohol 300mL, water 500mL are made into mixing solutions and are placed in separating funnel by normal hexane, ethyl acetate, methyl alcohol, water in the ratio of 4:5:3:5, leave 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 respectively 6mL equivalent upper and lower mutually in, ultrasonic 40min, fully dissolution sample and drain bubble.Setting constant temperature circulating actuator temperature is 35 ℃, opens UV-detector and N2000 chromatographic working station, and wavelength is set is 214nm.Start to pump into stationary phase with the flow velocity of 2mL/min, then slowly regulate flow velocity to 20mL/min, after stationary phase is full of pipeline, stop pumping into stationary phase, open main frame and turn speed to 800rpm, flow pump with 2mL/min enters moving phase, when having after moving phase outflow and baseline balance, by sampling valve sample introduction, record color atlas (Fig. 1), collect cut (10mL/ pipe) with run tank, detect each cut with high performance liquid chromatograph (Agilent 1260 type high performance liquid chromatographs).
(3) collection of target compound and purity testing
HPLC testing conditions is as follows: Chrom-Matrix C18(250mm × 4.6mm I.D, 5 μ m), 40 ℃ of column temperatures; Moving phase is the 2mM Ammonium bicarbonate food grade aqueous solution (A phase) and 2mM Ammonium bicarbonate food grade/40% water/60% acetonitrile (B phase).Flow velocity 1.0mL/min; Dual wavelength (203 and 215nm) detects, 40 ℃ of column temperatures.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 (Fig. 2) and peimisine reference substance (Fig. 3) are analyzed, show that the peak that in fritillaria total alkaloids, retention time is 17.8min is peimisine.
The collection of target compound: detect through the cut that run tank is collected successively, find that target compound (peimisine) flows out (Fig. 1) between 54 ~ 75 min 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%(Fig. 4 that high performance liquid chromatography area normalization method records purity), and molecular weight and characteristic ion have been confirmed it for peimisine (Fig. 5) in second order ms, solvent evaporated obtains peimisine 5.3mg.
Embodiment 2
A method of utilizing high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary, comprises the following steps:
(1) watt cloth fritillaria total alkaloids crude extract preparation
Get watt cloth bulb of fritillary meal that drying and crushing is good (crossing 20 mesh sieves) 100g, prepare total alkaloids by method in embodiment 1, obtain total alkaloids powder 578mg.
(2) high speed adverse current chromatogram separates
Application high speed adverse current chromatogram TBE-300B(Shanghai Tongtian Biotechnology Co., Ltd.) separation and purification peimisine.
High speed adverse current chromatogram two phase solvent system is normal hexane-ethyl acetate-methanol-water (3:5:3:5, v/v/v).The fritillaria total alkaloids powder 50mg that gets 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 embodiment 1.Purity is 97.2%, and solvent evaporated finally obtains peimisine 4.4mg.
Embodiment 3
Selection has the solvent system of suitable allocation coefficient, it is generally acknowledged that partition ratio K value separating effect between 0.5 ~ 2 is better.
The mensuration of K value: get bulb of fritillary alkaloid (about 1mg) in 4mL EP test tube, by the two-phase solvent system that reaches in advance partition equilibrium, upper and the lower mutually each 0.5mL that gets same volume is dissolved, thermal agitation fully mixes by it, separates after balance wait reaching, get respectively the upper of same volume with lower phase sample solution in two test tubes, dry up with nitrogen, respectively add 0.5mL chromatogram dissolve with methanol, carry out HPLC detection, peak area is designated as respectively A
on, A
under, partition ratio K is calculated as follows: K=A
on/ A
under, the results are shown in Table 1.
In methylene chloride-methanol-water system, the partition ratio recording is less than normal as can be known from Table 1, and visible solubleness in stationary phase is too little, when adverse current chromatogram separates, target compound is followed moving phase wash-out out, and appearance time is very short, does not have time enough to separate, and in the time that actual HSCCC separates, confirming does not have target sample well to be separated, and adds the system of hydrochloric acid (aq, 0.01M) yet, its partition ratio is bigger than normal, is also not suitable for as the solvent system separating.The present invention's discovery, in normal hexane-ethyl acetate-methanol-water solvent system, the partition ratio recording is comparatively suitable, therefore selects this system as the solvent systems that separates peimisine.By test, normal hexane-ethyl acetate-methanol-water (2 ~ 4:5:2 ~ 3:5) is compared the system of other ratio, can separate and obtain the target compound that purity is higher.
The partition ratio (K) of table 1 target component in different solvents system
Claims (3)
1. a method of utilizing high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary, is characterized in that, comprises the following steps:
1) get dry bulb of fritillary bulb, be ground into meal, add liquid ammonia alkalinization, with alcohol thermal backflow extraction, united extraction liquid, concentrating under reduced pressure obtains medicinal extract; By medicinal extract 0.1M dissolving with hydrochloric acid, filter to obtain acid solution, acid solution is first used to petroleum ether extraction degreasing, get sour water layer 10% sodium hydroxide and adjust pH to 9 ~ 10, then with dichloromethane extraction for several times, combined dichloromethane liquid, the dry fritillaria total alkaloids that to obtain of concentrating under reduced pressure;
2) adopt high speed adverse current chromatogram separation and purification peimisine; By normal hexane, ethyl acetate, methyl alcohol, water be by volume 2 ~ 4:5:2 ~ 3:5 be made into mixing solutions be placed in separating funnel leave standstill 12h; Get upper strata as stationary phase, lower floor is as moving phase; First pump into stationary phase, treat that stationary phase is full of high-speed counter-current chromatograph pillar, stop pumping into stationary phase, then pump into moving phase, when have moving phase to flow out and baseline balance after; get fritillaria total alkaloids powder and be dissolved in moving phase and the stationary phase of equivalent; by sampling valve sample introduction, observe color atlas, in the time that target peak occurs, collect cut; volatilize solvent, obtain peimisine.
2. the method for utilizing high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary claimed in claim 1, is characterized in that: in step 1), in described extracting method, the concentration of alcohol is 70%～95%.
3. the method for utilizing high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary claimed in claim 1, is characterized in that: step 2) in, high speed adverse current chromatogram is operating as: the constant temperature circulating actuator temperature of setting is 35 ℃, and the wavelength of setting is 214nm; The flow velocity that pumps into stationary phase is 5 ~ 20mL/min, and engine speed is 800 ~ 850rpm, and the flow velocity that pumps into moving phase is 1.5 ~ 2mL/min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114720576A (en) * | 2021-01-05 | 2022-07-08 | 中国科学院大连化学物理研究所 | Method for enriching and purifying steroid alkaloid in fritillaria medicinal material |
| CN115433254A (en) * | 2022-08-30 | 2022-12-06 | 四川大学 | Extraction method of fritillaria alkaloid component and application of fritillaria alkaloid component in bronchitis resistance |
| CN115448972A (en) * | 2022-08-30 | 2022-12-09 | 四川大学 | Extraction method of fritillaria alkaloid and application of fritillaria alkaloid in pulmonary fibrosis resistance |
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| CN101798307A (en) * | 2010-03-26 | 2010-08-11 | 巴塞利亚药业（中国）有限公司 | Preparation method of cyclopamine |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101798307A (en) * | 2010-03-26 | 2010-08-11 | 巴塞利亚药业（中国）有限公司 | Preparation method of cyclopamine |
Non-Patent Citations (2)
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| 叶耀辉 等: "贝母生物碱提取分离方法的研究进展", 《赣南医学院学报》 * |
| 张荣劲 等: "高速逆流色谱分离天然产物的溶剂体系选择", 《中草药》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114720576A (en) * | 2021-01-05 | 2022-07-08 | 中国科学院大连化学物理研究所 | Method for enriching and purifying steroid alkaloid in fritillaria medicinal material |
| CN114720576B (en) * | 2021-01-05 | 2024-01-23 | 中国科学院大连化学物理研究所 | Method for enriching and purifying steroid alkaloids in fritillaria medicinal materials |
| CN115433254A (en) * | 2022-08-30 | 2022-12-06 | 四川大学 | Extraction method of fritillaria alkaloid component and application of fritillaria alkaloid component in bronchitis resistance |
| CN115448972A (en) * | 2022-08-30 | 2022-12-09 | 四川大学 | Extraction method of fritillaria alkaloid and application of fritillaria alkaloid in pulmonary fibrosis resistance |
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