CN101293872B - Method for preparing high-purity laurotetanine with high speed reversed flow color spectrum method - Google Patents
Method for preparing high-purity laurotetanine with high speed reversed flow color spectrum method Download PDFInfo
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- CN101293872B CN101293872B CN2007100401666A CN200710040166A CN101293872B CN 101293872 B CN101293872 B CN 101293872B CN 2007100401666 A CN2007100401666 A CN 2007100401666A CN 200710040166 A CN200710040166 A CN 200710040166A CN 101293872 B CN101293872 B CN 101293872B
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Abstract
The invention discloses a method for preparing high-purity laurotetanine by high speed counter-current chromatography, comprising the steps of: preparing a solvent system for composing a stationary phase and a mobile phase; adjusting flow rate of the mobile phase; and adjusting the rotation speed of a motor. The method is characterized in that the solvent system is composed of chloroform, methanol and water with a volumetric ratio of (2-4):3:(2-2.5), the upper layer of solvent system is the stationary phase, and the lower layer of the solvent system is the mobile phase. The method has the advantages of mild separation conditions, high separation efficiency, short separation time, and high purity laurotetanine.
Description
Technical field
The present invention relates to prepare the method for high purity laurotetanine with high-speed countercurrent chromatography (HSCCC).
Background technology
Root of Mountain Spicy Tree is the root and rhizome of Lauraceae Litsea plant Tetranthera citrata (Litsea cubeba (Lour.) Pers).Has expelling wind and cold, effects such as regulating QI to relieve pain.Wherein contained alkaloid laurotetanine is a compound that pharmacologically active is strong, has the effect of the excited spinal cord of class Strychnine.
Laurotetanine is extremely low at the occurring in nature amount, and contains a large amount of impurity, therefore has a large amount of researchists exploring the ideal separation purification method so far.The method of the extraction laurotetanine that people such as Masao report in document Studies on theAlkaloids of Formosan Lauraceous Plants.V.Alkaloids of Litsea cubebaPersoon is as follows: Root of Mountain Spicy Tree is used extraction using alcohol for 22.38 kilograms, ethanol extraction 3% acetic acid extraction, be concentrated into certain volume place one night after-filtration, filtrate is used chloroform extraction, acid mother liquid is neutralized to ammonium hydroxide and separates out solid after extracting, come out with chloroform extraction, extracting solution extracts with 2% sodium hydroxide solution jolting again, sodium hydroxide extraction liquid wherein adds volatile salt to separating out crystallization, use chloroform extraction again, obtain the brown residue with the Anhydrous potassium carbonate drying.The acetone soln of residue adds picrolonate and gets red crystallization, filter, with ethanol repeatedly recrystallization obtain laurotetanine 7.7 grams, yield only is 0.034%.But this method complex operation step is introduced soda acid and recrystallization repeatedly, repeatedly causes target compound chemical transformation and physical change, lose greatly in the process, thereby the rate of recovery is low.
High-speed countercurrent chromatography (HSCCC) be a kind of successive that developed recently gets up need not any solid support efficiently, liquid liquid distribution chromatography isolation technique fast, its principle is to utilize one-way fluid kinetic balance phenomenon to realize the high-speed separation of compound.In such dynamic equilibrium system, two kinds of immiscible solvent phase uniaxially in rotating spiral tube distributes.During high-speed counter-current chromatograph work, the spiral tube in the chromatographic instrument is done planetary motion, because the effect of gravity and spiral tube power, fixed phase drift makes stationary phase be kept to the end of going into of spiral tube, and two-phase solvent is mixed in spiral tube simultaneously.Because the partition ratio difference of different solutes in two-phase, solute carries out partition equilibrium in two-phase solvent, thereby heterogeneity is separated.Operation is simple for high-speed countercurrent chromatography, need not sample is carried out complicated processing, and can with highly sensitive detection technique coupling, convenient and swift accurately.This technology is of wide application, and it can be arbitrarily that the two phase solvent system of using among the HSCCC is formed, so go for the separation of all cpds.It need not solid carrier HSCCC and all can realize preferably circulation ratio through repeated isolation repeatedly and obtain the higher rate of recovery.The product purity height is selected correct solvent system, can will obtain being higher than the product of 90% purity after the sample separation, is applicable to that the preparation type separates.
But desire selects the The suitable solvent system extremely important with the separation that HSCCC carries out success.Different solvent systemss, the ratio with different upper and lower phases, character such as its viscosity, polarity, density differ greatly, and identical composition is had different dissolvings, distribution capability, form the difference of partition ratio, and separating effect is produced different influences.At present, the selection of solvent system does not also have the complete theoretical foundation of a cover.In addition, after having selected solvent system, need sometimes three instrument operating parameters (rotating speed, flow rate of mobile phase, sampling volume) are set.
Summary of the invention
The technical problem to be solved in the present invention is the defective that overcomes traditional isolation technique, adopts high-speed countercurrent chromatography (HSCCC) to prepare the method for high purity laurotetanine.This method separation condition gentleness, the separation efficiency height, and disengaging time is short, can obtain highly purified laurotetanine.
Technical scheme of the present invention is as follows:
High-speed countercurrent chromatography prepares the method for high purity laurotetanine, comprising: preparation constitutes the solvent system of stationary phase and moving phase; Regulate flow rate of mobile phase; Regulate engine speed; It is characterized in that described solvent system is made up of chloroform, first alcohol and water, the volume ratio of three kinds of components is 2～4:3:2～2.5, is stationary phase mutually on this solvent system, is moving phase mutually down.
Do not destroying under the adjusting of system equilibrated, can regulate chloroform arbitrarily in above-mentioned scope: methyl alcohol: the volume ratio of water, preferred volume ratio is 4:3:2 or 4:3:2.5 or 3.5:3:2.
Engine speed must be controlled in 1.5～2.5ml/min scope, preferred 2.0ml/min; The flow velocity of moving phase must be controlled in 750～880rpm scope, preferred 800rpm; The temperature of constant temperature circulator is arranged in 23～27 ℃, preferred 25 ℃.
Concrete operations step of the present invention is as follows:
Root of Mountain Spicy Tree ethanol extraction n-butanol extraction, n-butanol extraction is partly used the silicagel column rough segmentation once, obtain the mixture of enrichment laurotetanine, accurately take by weighing the mixture of enrichment laurotetanine, use the moving phase sample dissolution, pump into moving phase and stationary phase simultaneously with the double pump equal proportion, when solvent when pipeline outlet is flowed out, stop to pump into stationary phase, set flow rate of mobile phase, start main motor simultaneously, regulate rotating speed, after tens minutes, the tubing string internal solvent is reached running balance, and the tubing string exit has only moving phase to flow out.Inject sample by sampler, the wavelength of setting detector is 280nm, the laurotetanine peak occurs in about 4 hours to 6 hours behind sample introduction, collects this stream part, obtains pure laurotetanine monomer after concentrating.
Owing to adopted processing condition such as rational solvent system, main control system rotating speed and flow rate of mobile phase, can obtain highly purified laurotetanine with the inventive method.And this separation and purification process condition gentleness, disengaging time is short, owing to do not introduce soda acid, thereby has reduced the variation of compound chemistry character, keeps the stable of compound and reduces the loss, and compound does not run off and does not have dead absorption yet.
Embodiment
Embodiment 1
Root of Mountain Spicy Tree is used extraction using alcohol for 5 kilograms, and ethanol extraction n-butanol extraction, n-butanol extraction are partly used the silicagel column rough segmentation once, obtains mixture 15 grams of enrichment laurotetanine.
Accurately take by weighing mixture 3.0 grams of enrichment laurotetanine, use chloroform: methyl alcohol: the following phased soln sample of water (4:3:2) solvent systems, open constant temperature circulator, it is 25 ℃ that temperature is set, being stationary phase mutually on this solvent systems, be moving phase mutually down, pump into moving phase and stationary phase simultaneously with the double pump equal proportion, when solvent when pipeline outlet is flowed out, stop to pump into stationary phase, the setting flow rate of mobile phase is 2ml/min, start main motor simultaneously, the adjusting rotating speed is 800rpm, after tens minutes, the tubing string internal solvent is reached running balance, and the tubing string exit has only moving phase to flow out.Inject sample by sampler, the setting wavelength is 280nm, occurs the laurotetanine peak behind sample introduction in about 4 hours 40 minutes, collect this stream part, obtain pure laurotetanine 1.4 grams after concentrating, its moderate purity is greater than 99.5%, yield 46.7% before the relative sample introduction, the medicinal material yield is 0.138% relatively.
Embodiment 2
Accurately take by weighing mixture 2.4 grams of enrichment laurotetanine among the embodiment 1, use chloroform: methyl alcohol: the following phased soln sample of water (3.5:3:2) solvent systems, open constant temperature circulator, it is 26 ℃ that temperature is set, being stationary phase mutually on this solvent systems, be moving phase mutually down, pump into moving phase and stationary phase simultaneously with the double pump equal proportion, when solvent when pipeline outlet is flowed out, stop to pump into stationary phase, the setting flow rate of mobile phase is 1.5ml/min, start main motor simultaneously, the adjusting rotating speed is 750rpm, after tens minutes, the tubing string internal solvent is reached running balance, and the tubing string exit has only moving phase to flow out.Inject sample by sampler, the setting wavelength is 280nm, occurs the laurotetanine peak behind sample introduction in about 5 hours 35 minutes, collect this stream part, obtain pure laurotetanine 1.0 grams after concentrating, its purity is greater than 99.5%, yield 40.4% before the relative sample introduction, the medicinal material yield is 0.121% relatively.
Embodiment 3
Accurately take by weighing mixture 2.6 grams of enrichment laurotetanine among the embodiment 1, use chloroform: methyl alcohol: the following phased soln sample of water (4:3:2.5) solvent systems, open constant temperature circulator, it is 27 ℃ that temperature is set, being stationary phase mutually on this solvent systems, be moving phase mutually down, pump into moving phase and stationary phase simultaneously with the double pump equal proportion, when solvent when pipeline outlet is flowed out, stop to pump into stationary phase, the setting flow rate of mobile phase is 2.5ml/min, start main motor simultaneously, the adjusting rotating speed is 880rpm, after tens minutes, the tubing string internal solvent is reached running balance, and the tubing string exit has only moving phase to flow out.Inject sample by sampler, the setting wavelength is 280nm, the laurotetanine peak occurs in about 5 hours behind sample introduction, collects this stream part, obtains pure laurotetanine 1.1 grams after concentrating.Its purity is greater than 99.5%, yield 42.3% before the sample introduction relatively, and the medicinal material yield is 0.127% relatively.
Embodiment 4
Accurately take by weighing mixture 1.2 grams of enrichment laurotetanine among the embodiment 1, use chloroform: methyl alcohol: the following phased soln sample of water (2:3:2) solvent systems, open constant temperature circulator, it is 24 ℃ that temperature is set, being stationary phase mutually on this solvent systems, be moving phase mutually down, pump into moving phase and stationary phase simultaneously with the double pump equal proportion, when solvent when pipeline outlet is flowed out, stop to pump into stationary phase, the setting flow rate of mobile phase is 2ml/min, start main motor simultaneously, the adjusting rotating speed is 800rpm, after tens minutes, the tubing string internal solvent is reached running balance, and the tubing string exit has only moving phase to flow out.Inject sample by sampler, the setting wavelength is 280nm, occurs the laurotetanine peak behind sample introduction in about 5 hours 30 minutes, collect this stream part, obtain pure laurotetanine 0.5 gram after concentrating, its purity is greater than 99.5%, yield 41.7% before the relative sample introduction, the medicinal material yield is 0.125% relatively.
Embodiment 5
Accurately take by weighing mixture 1.4 grams of enrichment laurotetanine among the embodiment 1, use chloroform: methyl alcohol: the following phased soln sample of water (3:3:2.5) solvent systems, open constant temperature circulator, it is 23 ℃ that temperature is set, being stationary phase mutually on this solvent systems, be moving phase mutually down, pump into moving phase and stationary phase simultaneously with the double pump equal proportion, when solvent when pipeline outlet is flowed out, stop to pump into stationary phase, the setting flow rate of mobile phase is 2.5ml/min, start main motor simultaneously, the adjusting rotating speed is 850rpm, after tens minutes, the tubing string internal solvent is reached running balance, and the tubing string exit has only moving phase to flow out.Inject sample by sampler, the setting wavelength is 280nm, the laurotetanine peak occurs in about 5 hours behind sample introduction, collects this stream part, obtains pure laurotetanine 0.6 gram after concentrating.Its purity is greater than 99.5%, yield 42.9% before the sample introduction relatively, and the medicinal material yield is 0.129% relatively.
Embodiment 6
Accurately take by weighing mixture 1.0 grams of enrichment laurotetanine among the embodiment 1, use chloroform: methyl alcohol: the following phased soln sample of water (1.5:1:3) solvent systems, open constant temperature circulator, it is 25 ℃ that temperature is set, being stationary phase mutually on this solvent systems, be moving phase mutually down, pump into moving phase and stationary phase simultaneously with the double pump equal proportion, when solvent when pipeline outlet is flowed out, stop to pump into stationary phase, the setting flow rate of mobile phase is 2.0ml/min, start main motor simultaneously, the adjusting rotating speed is 800rpm, after tens minutes, the tubing string internal solvent is reached running balance, and the tubing string exit has only moving phase to flow out.Inject sample by sampler, the setting wavelength is 280nm, the laurotetanine peak occurs in about 3 hours behind sample introduction, but overlaps with impurity peaks, and resolution is little, collects this stream part, obtains pure laurotetanine 0.2 gram after concentrating.Its purity is 80%, yield 20% before the relative sample introduction, and the medicinal material yield is 0.06% relatively.
Claims (1)
1. high-speed countercurrent chromatography prepares the method for high purity laurotetanine, comprising: preparation constitutes the solvent system of stationary phase and moving phase; Regulate flow rate of mobile phase; Regulate engine speed; It is characterized in that described solvent system is made up of chloroform, first alcohol and water, the volume ratio of three kinds of components is 3.5: 3: 2, be stationary phase mutually on this solvent system, be moving phase mutually down, the flow velocity of described moving phase is 1.5ml/min, and described engine speed is 750rpm.
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| 禇建军 等.高速逆流色谱在生物碱分离当中的应用.浙江化工34 8.2003,34(8),30-31. |
| 禇建军 等.高速逆流色谱在生物碱分离当中的应用.浙江化工34 8.2003,34(8),30-31. * |
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