CN103772472B - A kind of method utilizing high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary - Google Patents
A kind of method utilizing high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary Download PDFInfo
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- CN103772472B CN103772472B CN201410005295.1A CN201410005295A CN103772472B CN 103772472 B CN103772472 B CN 103772472B CN 201410005295 A CN201410005295 A CN 201410005295A CN 103772472 B CN103772472 B CN 103772472B
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Abstract
The invention discloses a kind of method utilizing high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary, comprising: 1) get dry bulb of fritillary bulb, prepare fritillaria total alkaloids; 2) high speed adverse current chromatogram separation and purification peimisine is adopted; Be that the mixing solutions that is made into of 2 ~ 4:5:2 ~ 3:5 is as stationary phase (upper strata) and moving phase (lower floor) by volume using normal hexane, ethyl acetate, methyl alcohol, water; Get moving phase and stationary phase that fritillaria total alkaloids powder is dissolved in equivalent, by sampling valve sample introduction, observe color atlas, when target peak occurs, collect cut, volatilize solvent, obtain peimisine.The present invention does not need to use solid phase carrier, and without irreversible adsorption, method is simple, fast, efficient, gained target compound purity is high.
Description
Technical field
The invention belongs to Natural Medicine Chemistry technical field, relate to a kind of method preparing peimisine from Traditional Chinese medicine fritillary, particularly one utilizes the method for high-speed countercurrent chromatography (HSCCC) separation and purification peimisine from the bulb of fritillary.
Background technology
Jie Li Lu class different tetrahydroisoquinoline alkaloid cyclopamine (cyclopamine) found 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, to kinds cancer as nonsmall-cell lung cancer, cancer of the stomach, carcinoma of the pancreas etc. have good inside and outside restraining effect.Because cyclopamine content in plant is lower, simultaneously chemistry is complete synthesis very difficult, from plant separating ring bar amine analogue, be comparatively key tactics by the semi-synthetic cyclopamine that obtains again.Peimisine (peimisine) is successfully changed into cyclopamine by existing people at present.Therefore, peimisine is a kind of compound having value of exploiting and utilizing.
The bulb of fritillary is relieving cough and reducing sputum conventional Chinese medicine, derives from the bulb of Liliaceae (Liliaceae) Fritillaria (Fritillaria) the multiple bulb of fritillary.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 bulb of fritillary is all containing 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, has 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, namely utilize one in immiscible two phase solvent system as the stationary phase of chromatographic separation, 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 techniques, do not exist sample irreversible adsorption and on impacts such as the pollutions of sample.It also has the series of advantages such as preparation amount is large, sepn process is short, sample pretreatment is simple, save solvent, economical and effective, solvent system selection scope are large compared with traditional column chromatography.Current high-speed countercurrent chromatography is widely used in the fields such as medication chemistry, in the isolation and purification process of natural product, be especially considered to be a kind of novel effective isolation technique.And suitable solvent systems (moving phase, stationary phase), the isoparametric optimization of rotating speed, flow velocity, sample size high speed adverse current chromatogram preparation in play critical effect, particularly in sample the partition ratio K value of each component whether suitable decide solvent systems, 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 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 the higher peimisine monomer of purity can be obtained.
Technical scheme: in order to realize foregoing invention object, the technical solution used in the present invention is:
Utilize a method for high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary, comprise the following steps:
1) get dry bulb of fritillary bulb, be ground into meal, add liquid ammonia alkalinization, use alcohol circumfluence distillation, united extraction liquid, concentrating under reduced pressure obtains medicinal extract; Medicinal extract is used 0.1M dissolving with hydrochloric acid, filters to obtain acid solution, acid solution is first used 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, concentrating under reduced pressure dry fritillaria total alkaloids;
2) high speed adverse current chromatogram separation and purification peimisine is adopted; 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, to be fixedly be full of high-speed counter-current chromatograph pillar mutually, stopping pumping into stationary phase, then pump into moving phase, to flow out when there being moving phase and after baseline balance; get moving phase and stationary phase that fritillaria total alkaloids powder is dissolved in equivalent; by sampling valve sample introduction, observe color atlas, when 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 DEG C, and the wavelength of setting is 214nm; The flow velocity pumping 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 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 higher peimisine monomer of purity.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
Utilize a method for high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary, 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.By medicinal extract 50mL 0.1M 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(Shanghai Tongtian Biotechnology Co., Ltd.) 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 (Fig. 1), collect cut (10mL/ pipe) with run tank, detect each cut with high performance liquid chromatograph (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 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, 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 (Fig. 2) and peimisine reference substance (Fig. 3) 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 (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 confirming it for peimisine (Fig. 5) through second order ms middle-molecular-weihydroxyethyl and characteristic ionic, solvent evaporated obtains peimisine 5.3mg.
Embodiment 2
Utilize a method for high-speed countercurrent chromatography separation and purification peimisine from the bulb of fritillary, comprise the following steps:
(1) watt cloth fritillaria total alkaloids crude extract preparation
Get the good watt cloth bulb of fritillary meal of drying and crushing (crossing 20 mesh sieves) 100g, prepare total alkaloids by method in embodiment 1, obtain total alkaloids powder 578mg.
(2) high speed adverse current chromatogram is separated
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 n-hexane-ethyl acetate-methanol-water (3:5:3:5, 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 embodiment 1.Purity is 97.2%, and solvent evaporated finally obtains peimisine 4.4mg.
Embodiment 3
Select the solvent system with 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 reaching partition equilibrium in advance, get the upper of same volume and lower mutually each 0.5mL is dissolved, thermal agitation fully mixes by it, after separation to be achieved balance, gets the upper of same volume respectively 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 A respectively
on, A
under, partition ratio K is then calculated as follows: K=A
on/ A
under, the results are shown in Table 1.
As can be known from Table 1 in methylene chloride-methanol-water system, the partition ratio recorded is less than normal, and the solubleness as seen in stationary phase is too little, when adverse current chromatogram is separated, with moving phase wash-out out, appearance time is very short for target compound, does not have time enough to be separated, and when actual HSCCC is separated, also confirm do not have target sample well to be separated, and add the system of hydrochloric acid (aq, 0.01M), its partition ratio is bigger than normal, is also not suitable for the solvent system as being separated.The present invention finds, in n-hexane-ethyl acetate-methanol-water solvent system, the partition ratio recorded is comparatively suitable, therefore selects this system as the solvent systems being separated peimisine.By test, n-hexane-ethyl acetate-methanol-water (2 ~ 4:5:2 ~ 3:5) compares the system of other ratio, can be separated and obtain the higher target compound of purity.
The partition ratio of table 1 target component in different solvents system (K)
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.
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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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| 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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| 贝母生物碱提取分离方法的研究进展;叶耀辉 等;《赣南医学院学报》;20130831;第33卷(第4期);第632页右栏倒数第2段至第633页左栏第1段 * |
| 高速逆流色谱分离天然产物的溶剂体系选择;张荣劲 等;《中草药》;20080212;第39卷(第2期);第299页1.3.1，第302页左栏3.2 * |
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