CN100422187C - New method for extracting cephalotaxine and percephalotaxine - Google Patents
New method for extracting cephalotaxine and percephalotaxine Download PDFInfo
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- CN100422187C CN100422187C CNB021503230A CN02150323A CN100422187C CN 100422187 C CN100422187 C CN 100422187C CN B021503230 A CNB021503230 A CN B021503230A CN 02150323 A CN02150323 A CN 02150323A CN 100422187 C CN100422187 C CN 100422187C
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- harringtonine
- homoharringtonine
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- Y02P20/00—Technologies relating to chemical industry
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Abstract
The present invention provides a method for simplifying production technology, rationally using resources of rare wood species, decreasing emission of waste and effectively improving the yield of preparations. The present invention adopts reproducible cephalotaxus fortunei small leaves and branches with low contents of harringtonine and homoharringtonine, and comprises the following steps of chalking with alkalization by 10% of dilute ammonia water, supercritical CO2 fluid extraction, separation and recovery of cephalotaxus fortunei total alkaloids, continuous column chromatography by adsorptive resin, recovery of cephalotaxus fortunei mixed ester alkali, preparation of high-speed countercurrent chromatography and ultraviolet detection to obtain monomers of harringtonine and homoharringtonine and a small quantity of mixtures of the harringtonine and the homoharringtionine, and respective recrystallization and purification to obtain pure products used in the preparations.
Description
Technical field
The present invention relates to a kind of extracting method of biotic component, particularly relate to a kind of natural anti-cancer drugs---the extracting method of harringtonine and homoharringtonine.
Background technology
Along with the continuous development of science and technology, people have found the method for the treatment of to many diseases of incurable disease that are originally.A kind of extremely have rare seeds a---Cephalotaxus fortunei (CephalotaxusLanceolata K.M.Feng), measure through clinical medicine, the alkaloid that its bark and other positions contain---harringtonine and homoharringtonine, be natural antileukemie good medicine, the appearance of this biotechnological formulation, the hope of having brought life for many amort leukaemics.
Traditional extraction process is to be raw material with the Cephalotaxus fortunei bark, takes the alcohol diacolation, recovered alcohol, and through acid-alkali treatment repeatedly repeatedly, chloroform extraction, again through the neutral alumina adsorpting column chromatography, the partition column chromatography of silica gel obtains harringtonine and homoharringtonine.
The weak point of aforesaid method is: complex manufacturing, and the cycle is long, poor operability, turnout is minimum, and environmental pollution is more serious, and the rate of recovery is low, can't realize the industrialization operate continuously, and greatly destroys the rare tree species resource, is unfavorable for preserving the ecological environment.
Patent Office of the People's Republic of China has announced that application number is the invention (applying date is 1999.03.17) of 99806044.5 " new cephalotaxane derivative and preparation method thereof "; this Invention Announce the mode of the natural harringtonine of a kind of direct acidylate of acylate that adopts side chain precursor to constitute, a kind of method that generates harringtonine by chemosynthesis is provided.
This method cost of investment is bigger, and still need adopt natural Cephalotaxin as raw material, has the shortcoming of aforementioned conventional technology equally.
Summary of the invention
The object of the present invention is to provide and a kind ofly can simplify production technique, rationally used the rare tree species resource, reduced waste discharge, and can effectively improve the method for preparation output.
In order to achieve the above object, the little branches and leaves of reproducible Cephalotaxus fortunei that the present invention adopts harringtonine and homoharringtonine content to be lower than the Cephalotaxus fortunei bark are raw material, are prepared from following machining process: beat powder also with 10% weak ammonia alkalization → supercritical CO
2The continuous column chromatography of fluid extraction → Separation and Recovery cephalotaxus fortunei total alkaloids → polymeric adsorbent → recovery Cephalotaxus fortunei mixed ester alkali → preparation high speed adverse current chromatogram also obtains the recrystallization purifying of harringtonine and homoharringtonine monomer and a spot of the two mixture → respectively through ultraviolet detection and obtains the pure product used for preparation.
It is 0.5-3mm that described raw material is beaten powder powder degree, and solid accumulation density is 228kg/m
3
Described supercritical CO
2Fluid is entrainment agent with ethanol, and flow is 180-190kg/h;
Described supercritical CO
2The processing condition of fluid extraction are: the extraction time is 40 ℃ for the 3h extraction temperature, and extracting pressure is 20MPa;
The continuous column chromatography of described polymeric adsorbent is made up of four processes: a macroporous adsorbent resin column chromatography → primary ions exchange column chromatography → secondary macroporous adsorbent resin column chromatography → secondary ions exchange column chromatography.
Described macroporous adsorbent resin is the AB8 macroporous adsorbent resin, and it is citric acid-methanol system of 6% that macroporous adsorbent resin column chromatography adopts strippant, and the sample adsorptive capacity is by dried cream and resin 1: the calculating of 15-20 ratio, flow velocity is 0.5-5ml/cm
2/ min; It is that pH value is 5.0 Sodium phosphate dibasics-citrate buffer solution and methanol system that the secondary macroporous adsorbent resin column chromatography adopts strippant, and the sample adsorptive capacity is in dried cream and resin 1: the calculating of 10-15 ratio, flow velocity is 0.5-5ml/cm
2/ min.
Described ion exchange resin is 100
*The 7# Zeo-karb, the strippant that primary ions exchange column chromatography is adopted is the sodium hydroxide and the methanol system of different pH concentration, and the sample exchange capacity is in dried cream and 1: 20 ratio of resin, and flow velocity is 0.5-3ml/cm
2/ min; The strippant that secondary ions exchange resin column chromatography adopts is that the pH value is sodium hydroxide and the methanol system more than 9.0, and the sample exchange capacity is in dried cream and 1: 20 ratio of resin, and flow velocity is 0.5-3ml/cm
2/ min.
Described high speed adverse current chromatogram prepares as follows: column volume is 1000ml, solvent systems is: chloroform, 0.07mol/L the pH value that sodium phosphate-0.04mol/L citric acid is formed is 5.0 damping fluid, retention value 80%, rotating speed 800 commentaries on classics/min, flow velocity 200ml/h, the detection wavelength is 284nm, collects every pipe automatically and detects in per 2 minutes.
Owing to adopt above-mentioned technology, make the present invention have following advantage:
(1) used content low but can easily regenerate and resource replaces bark as raw material more than the abundant Cephalotaxus fortunei sprig leaf of bark, solved the rare problem of Cephalotaxus fortunei bark, protected species resource than bark;
(2) wide material sources and recoverable CO have been used
2Replace noxious solvent, can under the near ambient temperature condition, operate, be beneficial to the extraction of biologically active substance, and reduced processing step, reduce and pollute and energy consumption;
(3) use the technology of the continuous column chromatography of polymeric adsorbent, both can cut down the consumption of energy, realize the workerization continuous production, the discharging that can reduce organic solvent is arranged, the ecotope that protection is human, and polymeric adsorbent can regenerate and recycle, and greatly reduces production cost;
(4) adopt the adverse current chromatogram need not solid-state supporter, finish and got rid of supporter detrimentally affects such as the absorption of sample component, taint, sex change, inactivation, the very high rate of recovery of solute chromatographic peak conditions of streaking that can avoid irreversible adsorption to cause.
Production technique has been simplified in proposition of the present invention, has reduced the pollution to environment, has improved production efficiency, has satisfied the market requirement of growing natural anti-cancer drugs, has protected precious natural resources and ecotope, makes man and nature to develop in harmony.
Description of drawings
It shown in the accompanying drawing process flow sheet of the present invention.
Embodiment
Below in conjunction with accompanying drawing preferred forms of the present invention is described in further detail.
Raw material breaks into meal with crusher earlier, and the powder degree is 0.5-3mm.After pulverulent material alkalized with 10% weak ammonia, in the 1000 liters of charging baskets of packing into, tap density was 228kg/m
3
The charging basket that material is housed being hung in the extraction kettle, close and lock clip, will be the liquid CO of entrainment agent with 85% ethanol
2, after the preheater heating, squeeze into extraction kettle, material is carried out forward supercritical extraction, CO
2Flow is 180-190kg/h, and the extraction time is 3h, and extraction temperature is 40 ℃, and extracting pressure is 20MPa; Carry the supercutical fluid of effective constituent, after shut off valve decompression, cooling, enter separator, wherein effective ingredient is measured and is collected through high performance liquid chromatograph, promptly gets total alkaloids, CO
2Gas flows into withdrawing can after condenser liquefaction, use through the high-pressure pump pressurized circulation again.
Select AB8 macroporous adsorbent resin and 100
*The 7# Zeo-karb is the carrier of column chromatography, and resin need be anticipated, and with the ethanol continuous washing for several times, adding suitable quantity of water to washings does not have white opacity when (1ml ethanol liquid adds 5ml water), with a large amount of distilled water flush away ethanol, standby then.Sample solution is through precipitating in advance, and the pH value is regulated 7.5-8.0, filters, and ethanol wet method dress post is opened peristaltic pump, and dried cream and resin connect the last sample of ratio 1: 15-20, and as the elutriant desorb, flow velocity is 0.5-5ml/cm with 6% citric acid-methyl alcohol buffer solution system
2/ min measures with high performance liquid chromatograph.
Proceed the ion exchange resin column chromatography, with the Zeo-karb dress post of getting ready, dried cream and resin in proportion 1: the last sample of 10-15, the gradient eluent of making by different pH values with sodium hydroxide-methanol system continuously flows through the cylinder wash-out, and flow velocity is 0.5-3ml/cm
2/ min, Fractional Collections is measured with high performance liquid chromatograph.Select pH7-8 stream part, rotatory evaporator concentrates the sixth of volume, macroporous adsorptive resins on the second time, with the pH value is that 5.0 Sodium phosphate dibasic-citrate buffer solution and methanol system continuously flow through cylinder as elutriant, the sample adsorptive capacity is in the ratio 1 of dried cream and resin: the 10-15 ratio is calculated, and flow velocity still is 0.5-5ml/cm
2/ min measures with high performance liquid chromatograph.
Ion exchange resin column chromatography on the second time is dried cream and resin a sample on 1: 20 in proportion, is that the sodium hydroxide more than 9.0 and the elutriant of methanol system continuously flow through cylinder with the pH value, and flow velocity still is 0.5-3ml/cm
2/ min collects stream part, measures with high performance liquid chromatograph, and evaporate to dryness promptly gets mixed ester alkali.
The preparation high speed adverse current chromatogram, the selective solvent system is that the pH that chloroform, 0.07mol L sodium phosphate-0.04mol/L citric acid is formed is 5.0 damping fluids, the solvent that the two-phase of choosing is immiscible standing demix behind the shake well in separating funnel.Get as stationary phase as moving phase, earlier stationary phase is filled with tubing string, column volume is 1000ml, again the flow velocity of moving phase with 200ml/h pumped into, treat that moving phase flows out from the tubing string mouth, after the baseline stability, have sampler to inject in sample, sample size is 15ml, the velocity of rotation of pressing 800r/min this moment starts main frame, and the tubing string effluent detects through UV (ultraviolet ray), and the detection wavelength is 284mm, collect peak I, II, III, collected every pipe automatically and detect in per 2 minutes; After elutriant was collected 300ml, the reverse wash-out of following phase solvent by collecting peak a, b, c again as above-mentioned mode, promptly got harringtonine and homoharringtonine and a small amount of the two mixture.Two kinds of monomers can obtain the pure product of using for preparation through different solvent recrystallization purifying.By two kinds of monomers that above technology mode obtains, its purity can reach more than 98%, improves approximately about 3% than former technology purity, and the rate of recovery reaches 80%, has improved about 20% than former technology.
Claims (8)
1. method of extracting harringtonine and homoharringtonine, it is characterized in that: the little branches and leaves of reproducible Cephalotaxus fortunei that adopt harringtonine and homoharringtonine content to be lower than the Cephalotaxus fortunei bark are raw material, are prepared from following machining process: beat powder also with 10% weak ammonia alkalization → supercritical CO
2The continuous column chromatography of fluid extraction → Separation and Recovery cephalotaxus fortunei total alkaloids → polymeric adsorbent → recovery Cephalotaxus fortunei mixed ester alkali → preparation high speed adverse current chromatogram also obtains the recrystallization purifying of harringtonine and homoharringtonine monomer and a spot of the two mixture → respectively through ultraviolet detection and obtains the pure product used for preparation.
2. follow the extracting method according to described harringtonine of claim 1 and homoharringtonine, it is characterized in that, it is 0.5-3mm that raw material is beaten powder powder degree, and solid accumulation density is 228kg/m
3
3. the extracting method of harringtonine according to claim 1 and homoharringtonine is characterized in that supercritical CO
2Fluid ethanol in addition is an entrainment agent, and flow is 180-190kg/h.
4. the extracting method of harringtonine according to claim 1 and homoharringtonine is characterized in that supercritical CO
2The fluid extraction processing condition are: the extraction time is 3h, and extraction temperature is 40 ℃, and extracting pressure is 20MPa.
5. the extracting method of harringtonine according to claim 1 and homoharringtonine, it is characterized in that the continuous column chromatography of polymeric adsorbent is made up of four processes: a macroporous adsorbent resin column chromatography → primary ions resins exchange column chromatography → secondary macroporous adsorbent resin column chromatography → secondary ions exchange resin column chromatography.
6. the extracting method of harringtonine according to claim 5 and homoharringtonine, it is characterized in that, the macroporous adsorbent resin that is adopted is the AB8 macroporous adsorbent resin, it is 6% citric acid-methanol system that a macroporous adsorption resin chromatography adopts strippant, the sample adsorptive capacity is in dried cream and resin 1: the 15-20 ratio is calculated, and flow velocity is 0.5-5ml/cm
2/ min; It is that the pH value is 5.0 Sodium phosphate dibasics-citrate buffer solution and methanol system that the secondary macroporous adsorbent resin column chromatography adopts strippant, and dried cream of sample adsorptive capacity and resin are in 1: the 10-15 ratio is calculated, flow velocity 0.5-5ml/cm
2/ min.
7. the extracting method of harringtonine according to claim 5 and homoharringtonine is characterized in that the ion exchange resin that is adopted is 100
*It is the sodium hydroxide and the methanol system of different pH concentration that 7# Zeo-karb, primary ions exchange resin chromatography adopt strippant, and the sample exchange capacity is in dried cream and 1: 20 ratio of resin, and flow velocity is 0.5-3ml/cm
2/ min; The strippant that secondary ions exchange resin column chromatography adopts is that the pH value is sodium hydroxide and the methanol system more than 9.0, and the sample exchange capacity is in dried cream and 1: 20 ratio of resin, and flow velocity is 0.5-3ml/cm
2/ min.
8. the extracting method of harringtonine according to claim 1 and homoharringtonine, it is characterized in that, high speed adverse current chromatogram prepares as follows: column volume is 1000ml, solvent systems is: the pH value that chloroform, 0.07mol/L sodium phosphate-0.04mol/L citric acid are formed is 5.0 damping fluid, retention value 80%, rotating speed 800 commentaries on classics/min, flow velocity 200ml/h, the detection wavelength is 284nm, collects every pipe automatically and detects in per 2 minutes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021503230A CN100422187C (en) | 2002-11-01 | 2002-11-01 | New method for extracting cephalotaxine and percephalotaxine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021503230A CN100422187C (en) | 2002-11-01 | 2002-11-01 | New method for extracting cephalotaxine and percephalotaxine |
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| CN1493572A CN1493572A (en) | 2004-05-05 |
| CN100422187C true CN100422187C (en) | 2008-10-01 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161973B (en) * | 2011-01-26 | 2013-08-07 | 海南大学 | Method for preparing homoharringtonine and special strain |
| CN102351872A (en) * | 2011-10-08 | 2012-02-15 | 程泽志 | Method for extracting harringtonine |
| CN104402895B (en) * | 2014-12-12 | 2016-08-17 | 重庆泰濠制药有限公司 | A kind of purification process of homoharringtonine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4152214A (en) * | 1977-10-07 | 1979-05-01 | The United States Of America As Represented By The Secretary Of Agriculture | Production of homodeoxyharringtonine and other cephalotaxine esters by tissue culture |
| CN1340505A (en) * | 2000-09-01 | 2002-03-20 | 四川泰港生物科技(集团)股份有限公司 | Chromatographic process for separating taxusol by continuous distribution column |
-
2002
- 2002-11-01 CN CNB021503230A patent/CN100422187C/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4152214A (en) * | 1977-10-07 | 1979-05-01 | The United States Of America As Represented By The Secretary Of Agriculture | Production of homodeoxyharringtonine and other cephalotaxine esters by tissue culture |
| CN1340505A (en) * | 2000-09-01 | 2002-03-20 | 四川泰港生物科技(集团)股份有限公司 | Chromatographic process for separating taxusol by continuous distribution column |
Non-Patent Citations (8)
| Title |
|---|
| 三尖衫酯碱和高三尖衫酯碱的生物活性及临床应用. 卢大用等.天然产物研究与开发，第12卷第5期. 2000 |
| 三尖衫酯碱和高三尖衫酯碱的生物活性及临床应用. 卢大用等.天然产物研究与开发，第12卷第5期. 2000 * |
| 中国粗榧果实和枝叶中三尖衫酯碱和高三尖衫酯碱分离方法的研究. 何洪源.中草药，第32卷第3期. 2001 |
| 中国粗榧果实和枝叶中三尖衫酯碱和高三尖衫酯碱分离方法的研究. 何洪源.中草药，第32卷第3期. 2001 * |
| 抗肿瘤药物-三尖衫酯类碱的开发研究进展. 白雪芳等.中国生化药物杂志，第19卷第1期. 1998 |
| 抗肿瘤药物-三尖衫酯类碱的开发研究进展. 白雪芳等.中国生化药物杂志，第19卷第1期. 1998 * |
| 用改进逆流分配法分离三尖衫酯碱和高三尖衫酯碱. 李建辉.中国医院药学杂志，第18卷第1期. 1998 |
| 用改进逆流分配法分离三尖衫酯碱和高三尖衫酯碱. 李建辉.中国医院药学杂志，第18卷第1期. 1998 * |
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Address after: 355300 No. six, Lane eight, 615 West Road, Zherong County, Fujian Province Patentee after: FUJIAN COSUNTER PHARMACEUTICAL Co.,Ltd. Address before: 355300 No. six, Lane eight, 615 West Road, Zherong County, Fujian Province Patentee before: Cosunter Pharmaceutical Co. |
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