CN1142163C - Process for separating and purifying ebormycine from fermented myxobacterium liquid - Google Patents
Process for separating and purifying ebormycine from fermented myxobacterium liquid Download PDFInfo
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Abstract
The present invention discloses a method for separating and extracting Epothilone from fermentation liquid. The method for separating and extracting Epothilone from fermentation liquid separates and extracts the Epothilone from the fermentation liquid of myxobacteria by technical measures, such as mixed resin adsorption, solid-liquid fractional extraction, sieve chromatography, crystallization, high-efficiency liquid phase separation, etc., and the method for separating and extracting Epothilone from fermentation liquid obtains a highly purified tumor resisting compound. The extracting yield of the Epothilone A and B can reach more than 80%, the method for separating and extracting Epothilone from fermentation liquid reduces cost, enhances efficiency and greatly enhances the practicability and the magnifying property of industrial production, and the method for separating and extracting Epothilone from fermentation liquid also has good economic benefit.
Description
(1) technical field
The present invention relates to a kind of method of from the slime bacteria fermented liquid, separating the purifying tacrolimus compounds, relate in particular to a kind of method of from the slime bacteria fermented liquid, separating the purification ebormycine.
(2) background technology
The most successful clinically antineoplastic chemotherapy medicine is short microtubule polymerization class natural compounds taxol (paclitaxel, Taxol at present
) and analogue taxotere (docetaxel, Taxotere
), be used to the treatment of solid carcinomas such as ovarian cancer, thymic carcinoma, colorectal carcinoma, lung cancer and liver cancer.The success of induction type antitumor drug taxol and the deficiency in chemotherapy thereof (the cell resistance that occurs in low water solubility and the chemotherapy process etc.) impel the researchist further to screen the microtubule stabilizer with better chemical property, biological property and pharmacological property.Yet through long-time, a large amount of screenings, just found the new natural compounds of four classes---Yi Lu mycin (eleutherobin) up in recent years, base of a fruit Mycosporin (discodermolide), FilippoGammarelli mycin (1aulimalides) and ebormycine (Epothilone) have the microtubule stabilization function.Wherein, He Fule reports such as (H fle) separated 16 membered macrolide compounds ebormycine (Epothilone) A and the B that make new advances from slime bacteria sorangium cellulosum (Sorangium cellulosum) in 1993, nineteen ninety-five Bo Lage (Bollag) etc. finds the short microtubule polymerization activity of ebormycine in to the extensive screening of antineoplastic compound after, caused people's extensive concern.Similar with taxol, ebormycine can induce tubulin at low temperature with do not contain under GTP (guanosine triphosphate) (GTP) or microtubule-associated protein (MAPs) condition and form microtubule.Different with taxol is that ebormycine still keeps active to p-P-glycoprotein expression type multidrug resistance (MDR) cell strain system and tumour.In taxol sensitive cells strain system, epothilone B has bigger inhibition activity than ebomycin A or taxol.On the other hand, the ELA of taxol may cause non-blood side effect in clinical chemotherapy, and the ebormycine intracellular toxin signal pathway of trigger cell not.In addition, the studies show that microtubule stabilizer such as taxol or ebomycin A of tower Laski (Taraschi) etc. also have antimalarial active, can hinder the formation of merozoite.
Ebormycine makes people drop into huge enthusiasm and studies, in the hope of faster its exploitation being become antitumor drug than the simple structure of taxol, good water-solubility and great pharmaceutical potential.Chemists put into a lot of energy the synthetic of ebormycine and analogue thereof and separate, and still, for separate the purification ebormycine from fermented liquid, adopt Gothic people's such as (Gerth) method at present always.This method comprises resin absorption, the silicagel column gradient separations, and sieve chromatography separates four steps with high performance liquid phase.We studies show that, this method exists certain defective and deficiency, especially can't reach the various requirement of suitability for industrialized production.Mainly show: step is too loaded down with trivial details, makes that the efficient of operation is lower and the process expense is very high; The yield of product is lower, is no more than 30%, and process is lost up to 70%; During resin absorption, use Amberlite (Amberlite) XAD-16 resin, cost is very high, and the domestic resin substitute of still not having same model at present; Make ebomycin A and B reach medication purity (greater than 90%), must separate through high performance liquid phase, this is difficult to realize in industrial production.Through the retrieval of document and patent, do not have bigger progress and relevant patent in this direction both at home and abroad.
(3) summary of the invention
At above-mentioned defective, main purpose of the present invention is to provide a kind of method of separating the purification ebormycine from the slime bacteria fermented liquid.It can overcome the fraud section of existing procedure effectively, reaches the efficient and economic purpose of separating the purification ebormycine from the slime bacteria fermented liquid, thus the industrialization that realizes extracting flow process.
The objective of the invention is to be achieved through the following technical solutions, the concrete steps order is as follows:
(1) preparation of hybrid resin: with CD180, CAD-40, XDA, S-8, NKA-II, six kinds of resins of AB-8 mix by weight 0.8～1.2: 0.8～1.2: 1.8～2.2: 0.8～1.2: 2.8～3.2: 1.8～2.2, outstanding being dissolved in isopyknic distilled water is prepared into mixed resin solution;
(2) hybrid resin absorption: when carrying out the slime bacteria fermentation, the above-mentioned hybrid resin that adds 0.1%-5% (volume/volume) in liquid nutrient medium adsorbs, and sorbent material is present among the whole fermentation flow process, after the fermentation ends, filtration makes sedimentary polymeric adsorbent mixture, and is standby;
(3) solid-liquid stepwise solvent extraction: 1. use the methyl alcohol of 10-20 times of volume that the resin compound that step (2) obtains is extracted, be incubated 30-50 ℃, 24-48 hour, the centrifugal resin of removing obtained first part's extract; 2. with 40 ℃ of evaporated in vacuo of the methyl alcohol in first part's extract, pulverize extract, use the methylene dichloride of 10-20 times of volume to carry out the extraction of second step, be incubated 20-30 ℃, 24-48 hour, the centrifugal precipitation of removing, obtain the second section extract,, pulverize extract 3. with 30 ℃ of evaporated in vacuo of the methylene dichloride in the second section extract, use the normal hexane of 10-20 times of volume to carry out the extraction of the 3rd step, be incubated 20-30 ℃, 24-48 hour, centrifugal collecting precipitation, use the normal hexane flushing precipitation of 10% volume ratio, obtain the third part extract;
(4) sieve chromatography: above-mentioned third part extract heavily is dissolved in 2 times of volumes methanol, carries out dextrane gel (Sephadex LH-20) molecular sieve column chromatography with the application of sample amount of 2-5% column volume and separate; Moving phase is methyl alcohol or methylene dichloride, flow velocity be 0.5 column volume/hour, detect wavelength 254nm, the elution volume of ebormycine is a 1.5-2.5 times of column volume;
(5) crystallization: the collection specimen preparation of step (4) is become the ketone supersaturated solution, utilize the crystallizer preparation and collect crystallization; Method is that thermostatic bath is heated to 45-60 ℃, sample is put into crystallizing dish, be incubated 15-30 minute, all dissolve to sample, begin gradient cooling then, speed is 0.5～3 ℃/30 minutes, after being cooled to 35-40 ℃, use solvent evaporated method instead, constant temperature concentrated liquid volume is to 1/10th of initial volume, can obtain a large amount of white powder crystallizations in the crystallizing dish bottom, be the mixture of ebomycin A and B;
(6) high performance liquid phase separates: if obtain the ebomycin A or the B of complete purifying, can adopt high performance liquid phase to carry out ebomycin A and B mixture separation, use anti-phase semipreparative column (Hypersil ODS2 C18), specification is 250 * 8mm, packing material size 10 μ m, flow velocity is 2ml/min, 249nm detects, 24 ℃ of column temperatures, sample size 100 μ l, detection time 30min, moving phase is 65% methyl alcohol and 35% water; The residence time of ebomycin A is 9.6 minutes, and the residence time of epothilone B is 11.1 minutes; Can obtain the ebomycin A or the B of purifying after the collection.
The described CD180 of step (1) wherein, CAD-40, XDA, S-8, NKA-II, the weight ratio of six kinds of mixed with resin of AB-8 is 1: 1: 2: 1: 3: 2.
Wherein described absorption with the hybrid resin addition of step (2) is the 0.5%-2% of liquid nutrient medium volume ratio.
Wherein step (3) described 1. in, optimum extraction temperature is 35-40 ℃.
Wherein step (3) described 2. in, optimum extraction temperature is 22-25 ℃.
Wherein step (3) described 3. in, optimum extraction temperature is 25-28 ℃.
Wherein step (4) the suitableeest described application of sample amount is the 2.5-3.8% column volume.
The elution volume of the described ebormycine of step (4) wherein, when using methyl alcohol as moving phase, elution volume is a 1.8-2.3 times of column volume; When using methylene dichloride as moving phase, elution volume is a 1.5-2.1 times of column volume.
Wherein the described ketone supersaturated solution of step (5) is one or both in 2-butanone or the acetone.
Wherein the suitableeest speed of the described gradient cooling of step (5) is 0.5～1 ℃/30 minutes.
For separating ebomycin A and the B that purifies and prepare, measured mass spectrum (seeing accompanying drawing 4) respectively, uv atlas (seeing accompanying drawing 5), infrared spectrum (seeing accompanying drawing 6) and nmr spectrum (seeing accompanying drawing 7 and accompanying drawing 8) have been confirmed structure (seeing accompanying drawing 3).And anti-tumor activity (seeing Appendix 1) and the microtubule polymerization of having measured them promote active (seeing Appendix 2).
In the method for the invention, hybrid resin is 95-100% for the adsorption rate of ebormycine, and the extraction yield of ebormycine is 85-90% when finishing crystallisation step, purity is 95-97%, after finishing the high performance liquid phase separation, the extraction yield of ebomycin A and B is 82-89%, and purity is 99.9-99.99%.
Creativeness of the present invention is, developed low-cost, mixing and absorption resin efficiently, and its adsorption rate has substituted expensive import polymeric adsorbent up to 95-100%; Developed substep liquid-solid extraction technology, the silicagel column that substitutes in the former step is gradient elution separation, has improved final extraction yield greatly, has reduced cost; Developed crystallization processes, and developed special crystallizer, and creationaryly ebormycine is purified by crystallization, make that the extraction yield of ebomycin A and B is 85-90% when finishing crystallisation step, purity is up to 95-97%, reached the requirement that preparation becomes medicine fully.And in the original extraction flow process, carry out before high performance liquid phase separates, the purity less than 70% of ebormycine, can't directly prepare becomes medicine.Major advantage of the present invention is by technologies such as substep liquid-solid extraction and crystallizations, makes the extraction yield of antineoplastic compound ebormycine reach more than 80%, owing to used cheap hybrid resin to adsorb, greatly reduces cost, has improved efficient.When considering suitability for industrialized production, the drug effect of ebomycin A and B and toxicity are roughly suitable, can mix pharmacy, can form product so finish crystallising part, have improved the feasibility and the scalable property of suitability for industrialized production greatly.This invention has been started efficiently and the innovative technology of economic separation purification antineoplastic compound ebormycine, possesses the feasibility and the scalable property of suitability for industrialized production, has good economic benefits and application prospect.
(4) description of drawings 1. is separated the process flow sheet of purification ebormycine from the slime bacteria fermented liquid
Wherein: the configuration of 1 hybrid resin; The absorption of 2 hybrid resins; 3 solid-liquid stepwise solvent extractions; 4 sieve chromatographies; 5 crystallizations; 6 high performance liquid phase separate; The ebomycin A and the B of 7 complete purifying; The ebomycin A of 8 higher degrees and B mixed crystallization.Fig. 2. carry out the crystallizer organigram of crystallization operation
Wherein: 9 sample crystallizing dish; 10 thermometers; 11 sample cells; 12 sample cell sealing covers; 13 single-phase synchronous machines; 14 electric mixer controllers; 15 electric coupling transformers; 16 two position controllers; 17 power supplys; 18 agitators; 19 heating units; 20 temperature controllers; 21 distilled waters; 22 micro-vacuum pumps; 23 vacuum exhaust pipes.Fig. 3. the chemical structure of ebormycine (Epothilone) A and B
Shown in the figure, the R of ebomycin A is H; The R of epothilone B is CH
3Fig. 4. ebomycin A that purification obtains and the mass spectrogram of B
Shown in the figure, (4-A) sample of figure is the ebomycin A after purifying, and draws (M+H) of component
+Be 494.2561, the molecular formula of match compound is C
26H
39O
6NS, relative deviation 1.922e-06; (4-B) sample of figure is the epothilone B after purifying, (M+H) of component
+Be 508.2725, the molecular formula of match compound is C
27H
41O
6NS, relative deviation 4.942e-07, all the theoretical value with ebomycin A or B fits like a glove.Fig. 5. ebomycin A that purification obtains and the ultraviolet spectrogram of B
Shown in the figure, the ebomycin A that obtains of purifying has identical ultraviolet spectrogram with B, and the wavelength value and the molar extinction coefficient of characteristic peak are respectively λ
Max1=210, log ε
1=14.17, λ
Max2=249, log ε
2=3.97, fit like a glove with the theoretical value of ebomycin A or B.Fig. 6. the infrared spectrogram of the epothilone B that purification obtains
Shown in the figure, the infrared spectrogram of the epothilone B that purification obtains and the theoretical value of epothilone B fit like a glove.Fig. 7. ebomycin A that purification obtains and the PMR spectrogram of B
Shown in the figure, (7-A) sample of figure is the ebomycin A after purifying; (7-B) sample of figure is the epothilone B after purifying, and sample solution is deuterated methanol, and the theoretical value of result and ebomycin A or B fits like a glove.Fig. 8. ebomycin A that purification obtains and B's
13The C-NMR spectrogram
Shown in the figure, (8-A) sample of figure is the ebomycin A after purifying; (8-B) sample of figure is the epothilone B after purifying, and sample solution is deuterated methanol, and the theoretical value of result and ebomycin A or B fits like a glove.
Fig. 9. alternating temperature ultraviolet spectrophotometry detected result figure.
Annex 1: the anti tumor activity in vitro laboratory report of the ebomycin A after the purification and B
Conclusion shows: the ebomycin A after the purification during greater than 50ng/ml, can kill two kinds of tumour cells in concentration; Epothilone B after the purification during greater than 10ng/ml, can kill two kinds of tumour cells in concentration. Two samples all have Obvious anti tumor activity in vitro, and consistent with literature value. Annex 2: the short microtubule polymerization activity experiment report of the ebomycin A after the purification and B
Conclusion shows: the ebomycin A after the purification and B all can in the situation that does not have GTP, significantly promote little Again external polymerization of pipe, and the activity of B is higher than A, consistent with the document conclusion.
(5) embodiment embodiment 1: the present invention utilizes hybrid resin absorption, solid-liquid stepwise solvent extraction, sieve chromatography, crystallization and technique means such as high performance liquid phase separates, separation and Extraction ebormycine from a kind of slime bacteria sorangium cellulosum fermented liquid, the concrete steps order is as follows: the preparation of (1) hybrid resin: with CD180, CAD-40, XDA, S-8, NKA-II, six kinds of resins of AB-8 were by weight 1: 1: 2: mix at 1: 3: 2, outstanding being dissolved in isopyknic distilled water is prepared into mixed resin solution.(2) hybrid resin absorption: when carrying out the sorangium cellulosum fermentation, the hybrid resin that adds 2% (volume/volume) in the 2000ml liquid nutrient medium adsorbs, and sorbent material is present among the whole fermentation flow process, after the fermentation ends, filtration makes sedimentary polymeric adsorbent mixture, and is standby.(3) solid-liquid stepwise solvent extraction:
1. use the methyl alcohol of 15 times of volumes that the resin compound that step (2) obtains is extracted, be incubated 38 ℃, 36 hours, the centrifugal resin of removing obtained first part's extract;
2. with 40 ℃ of evaporated in vacuo of the methyl alcohol in first part's extract, pulverize extract, use the methylene dichloride of 15 times of volumes to carry out the extraction of second step, be incubated 24 ℃, 36 hours, the centrifugal precipitation of removing obtained the second section extract;
3. with 30 ℃ of evaporated in vacuo of the methylene dichloride in the second section extract, pulverize extract, use the normal hexane of 15 times of volumes to carry out the extraction of the 3rd step, be incubated 27 ℃, 36 hours, centrifugal collecting precipitation, use the normal hexane flushing precipitation of 10% volume ratio, obtain the third part extract; (4) sieve chromatography: above-mentioned third part extract heavily is dissolved in 2 times of volumes methanol, carries out dextrane gel (Sephadex LH-20) molecular sieve column chromatography with the application of sample amount of 3% column volume and separate; Moving phase is methyl alcohol, flow velocity be 0.5 column volume/hour, detect wavelength 254nm, the elution volume of ebormycine is 2.1 times of column volumes; (5) crystallization: the collection specimen preparation of step (4) is become the supersaturated solution of 2-butanone, utilize crystallizer (the crystallizer structure is seen accompanying drawing 2) preparation and collect crystallization; Method is that thermostatic bath is heated to 50 ℃, sample is put into crystallizing dish, be incubated 25 minutes, all dissolve to sample, begin gradient cooling then, speed is 0.8 ℃/30 minutes, after being cooled to 38 ℃, use solvent evaporated method instead, constant temperature concentrated liquid volume is to 1/10th of initial volume, can obtain a large amount of white powder crystallizations in the crystallizing dish bottom, be the mixture of ebomycin A and B; (6) high performance liquid phase separates: if obtain the ebomycin A or the B of complete purifying, can adopt high performance liquid phase to carry out ebomycin A and B mixture separation, use anti-phase semipreparative column (Hypersil ODS2 C18), specification is 250 * 8mm, packing material size 10 μ m, flow velocity is 2ml/min, 249nm detects, 24 ℃ of column temperatures, sample size 100 μ l, detection time 30min, moving phase is 65% methyl alcohol and 35% water; The residence time of ebomycin A is 9.6 minutes, and the residence time of epothilone B is 11.1 minutes; Can obtain the ebomycin A or the B of purifying after the collection.
In the method for the invention, hybrid resin is 100% for the adsorption rate of ebormycine, when finishing crystallisation step, the extraction yield of ebormycine is 90%, and purity is 97%, after finishing high performance liquid phase and separating, the extraction yield of ebomycin A and B is 89%, and purity is 99.99%.Embodiment 2: the present invention utilizes hybrid resin absorption, solid-liquid stepwise solvent extraction, sieve chromatography, crystallization and technique means such as high performance liquid phase separates, separation and Extraction ebormycine from a kind of slime bacteria sorangium cellulosum fermented liquid, the concrete steps order is as follows: the preparation of (1) hybrid resin: with CD180, CAD-40, XDA, S-8, NKA-II, six kinds of resins of AB-8 were by weight 0.8: 0.8: 1.8: mix at 0.8: 2.8: 1.8, outstanding being dissolved in isopyknic distilled water is prepared into mixed resin solution.(2) hybrid resin absorption: when carrying out the sorangium cellulosum fermentation, the hybrid resin that adds 1% (volume/volume) in the 1000ml liquid nutrient medium adsorbs, and sorbent material is present among the whole fermentation flow process, after the fermentation ends, filtration makes sedimentary polymeric adsorbent mixture, and is standby.(3) solid-liquid stepwise solvent extraction:
1. use the methyl alcohol of 10 times of volumes that the resin compound that step (2) obtains is extracted, be incubated 31 ℃, 46 hours, the centrifugal resin of removing obtained first part's extract;
2. with 40 ℃ of evaporated in vacuo of the methyl alcohol in first part's extract, pulverize extract, use the methylene dichloride of 10 times of volumes to carry out the extraction of second step, be incubated 20 ℃, 46 hours, the centrifugal precipitation of removing obtained the second section extract;
3. with 30 ℃ of evaporated in vacuo of the methylene dichloride in the second section extract, pulverize extract, use the normal hexane of 10 times of volumes to carry out the extraction of the 3rd step, be incubated 21 ℃, 46 hours, centrifugal collecting precipitation, use the normal hexane flushing precipitation of 10% volume ratio, obtain the third part extract; (4) sieve chromatography: above-mentioned third part extract heavily is dissolved in 2 times of volumes methanol, carries out dextrane gel (Sephadex LH-20) molecular sieve column chromatography with the application of sample amount of 2% column volume and separate; Moving phase is methylene dichloride, flow velocity be 0.5 column volume/hour, detect wavelength 254nm, the elution volume of ebormycine is 1.9 times of column volumes; (5) crystallization: the collection specimen preparation of step (4) is become the supersaturated solution of acetone, utilize crystallizer (the crystallizer structure is seen accompanying drawing 2) preparation and collect crystallization; Method is that thermostatic bath is heated to 45 ℃, sample is put into crystallizing dish, be incubated 30 minutes, all dissolve to sample, begin gradient cooling then, speed is 0.5 ℃/30 minutes, after being cooled to 36 ℃, use solvent evaporated method instead, constant temperature concentrated liquid volume is to 1/10th of initial volume, can obtain a large amount of white powder crystallizations in the crystallizing dish bottom, be the mixture of ebomycin A and B; (6) high performance liquid phase separates: if obtain the ebomycin A or the B of complete purifying, can adopt high performance liquid phase to carry out ebomycin A and B mixture separation, use anti-phase semipreparative column (Hypersil ODS2 C18), specification is 250 * 8mm, packing material size 10 μ m, flow velocity is 2ml/min, 249nm detects, 24 ℃ of column temperatures, sample size 100 μ l, detection time 30min, moving phase is 65% methyl alcohol and 35% water; The residence time of ebomycin A is 9.6 minutes, and the residence time of epothilone B is 11.1 minutes; Can obtain the ebomycin A or the B of purifying after the collection.
In the method for the invention, hybrid resin is 95% for the adsorption rate of ebormycine, when finishing crystallisation step, the extraction yield of ebormycine is 90%, and purity is 95%, after finishing high performance liquid phase and separating, the extraction yield of ebomycin A and B is 82%, and purity is 99.91%.Embodiment 3: the present invention utilizes hybrid resin absorption, solid-liquid stepwise solvent extraction, sieve chromatography, crystallization and technique means such as high performance liquid phase separates, separation and Extraction ebormycine from a kind of slime bacteria sorangium cellulosum fermented liquid, the concrete steps order is as follows: the preparation of (1) hybrid resin: with CD180, CAD-40, XDA, S-8, NKA-II, six kinds of resins of AB-8 were by weight 1.2: 1.2: 2.3: mix at 1.2: 3.2: 2.3, outstanding being dissolved in isopyknic distilled water is prepared into mixed resin solution.(2) hybrid resin absorption: when carrying out the sorangium cellulosum fermentation, the hybrid resin that adds 1% (volume/volume) in the 5000ml liquid nutrient medium adsorbs, and sorbent material is present among the whole fermentation flow process, after the fermentation ends, filtration makes sedimentary polymeric adsorbent mixture, and is standby.(3) solid-liquid stepwise solvent extraction:
1. use the methyl alcohol of 20 times of volumes that the resin compound that step (2) obtains is extracted, be incubated 50 ℃, 28 hours, the centrifugal resin of removing obtained first part's extract;
2. with 40 ℃ of evaporated in vacuo of the methyl alcohol in first part's extract, pulverize extract, use the methylene dichloride of 20 times of volumes to carry out the extraction of second step, be incubated 30 ℃, 26 hours, the centrifugal precipitation of removing obtained the second section extract;
3. with 30 ℃ of evaporated in vacuo of the methylene dichloride in the second section extract, pulverize extract, use the normal hexane of 20 times of volumes to carry out the extraction of the 3rd step, be incubated 30 ℃, 28 hours, centrifugal collecting precipitation, use the normal hexane flushing precipitation of 10% volume ratio, obtain the third part extract; (4) sieve chromatography: above-mentioned third part extract heavily is dissolved in 2 times of volumes methanol, carries out dextrane gel (Sephadex LH-20) molecular sieve column chromatography with the application of sample amount of 5% column volume and separate; Moving phase is methylene dichloride, flow velocity be 0.5 column volume/hour, detect wavelength 254nm, the elution volume of ebormycine is 2.1 times of column volumes; (5) crystallization: the collection specimen preparation of step (4) is become acetone and 2-butanone equal-volume blended supersaturated solution, utilize crystallizer (the crystallizer structure is seen accompanying drawing 2) preparation and collect crystallization; Method is that thermostatic bath is heated to 60 ℃, sample is put into crystallizing dish, be incubated 15 minutes, all dissolve to sample, begin gradient cooling then, speed is 1.5 ℃/30 minutes, after being cooled to 40 ℃, use solvent evaporated method instead, constant temperature concentrated liquid volume is to 1/10th of initial volume, can obtain a large amount of white powder crystallizations in the crystallizing dish bottom, be the mixture of ebomycin A and B; (6) high performance liquid phase separates: if obtain the ebomycin A or the B of complete purifying, can adopt high performance liquid phase to carry out ebomycin A and B mixture separation, use anti-phase semipreparative column (Hypersil ODS2 C18), specification is 250 * 8mm, packing material size 10 μ m, flow velocity is 2ml/min, 249nm detects, 24 ℃ of column temperatures, sample size 100 μ l, detection time 30min, moving phase is 65% methyl alcohol and 35% water; The residence time of ebomycin A is 9.6 minutes, and the residence time of epothilone B is 11.1 minutes; Can obtain the ebomycin A or the B of purifying after the collection.
In the method for the invention, hybrid resin is 99.5% for the adsorption rate of ebormycine, when finishing crystallisation step, the extraction yield of ebormycine is 90%, and purity is 97%, after finishing high performance liquid phase and separating, the extraction yield of ebomycin A and B is 89%, and purity is 99.97%.
Annex 1: the anti tumor activity in vitro laboratory report of the ebomycin A after the purification and B
The examining report book Sample title: (Epothilone) A of the Epothilones after the purification and B Censorship unit: position biotechnology National Key Laboratory of life institute of Shandong University Censorship people: Li Yuezhong Whether testing goal: detecting two samples can be at external kill tumor cell Hela and Bel-7402 Date received: 2001.5.29 reporting date: 2001.6.8 Sample: (1) buffer solution control sample; (2) ebomycin A; (3) epothilone B. Detection method: mtt assay Test results report is as follows: Test cell strain: Hela
Test cell strain: Bel-7402
| Sample concentration | 1000ng/ml | 100ng/ml | 50ng/ml | 10ng/ml | 5ng/ml | 1ng/ml |
| Sample (1) | - | - | - | - | - | - |
| Sample (2) | + | + | + | - | - | - |
| Sample (3) | + | - | + | - | + | - |
| Sample concentration | 1000ng/ml | 100ng/ml | 50ng/ml | 10ng/ml | 5ng/ml | 1ng/ml |
| Sample (1) | - | - | - | - | - | - |
| Sample (2) | + | + | - | - | - | - |
| Sample (3) | + | + | + | - | - | - |
Conclusion: (2) number sample during greater than 50ng/ml, can kill two kinds of tumour cells in concentration; (3) number sample is dense Degree can kill two kinds of tumour cells during greater than 10ng/ml. Two samples all have obvious anti tumor activity in vitro. Annex 2: the short microtubule polymerization activity experiment report of the ebomycin A after the purification and B
The examining report book Sample title: (Epothilone) A of the Epothilones after the purification and B Censorship unit: position biotechnology National Key Laboratory of life institute of Shandong University Censorship people: Li Yuezhong Testing goal: detect whether two samples can promote microtubule under no GTP condition polymerization in vitro Date received: 2001.6.2 reporting date: 2001.6.16 Sample: (1) buffer solution control sample; (2) ebomycin A; (3) epothilone B. Detection method: alternating temperature ultraviolet spectrophotometry Test results report as shown in Figure 9. Conclusion: (2) number and (3) number sample all can in the situation that does not have GTP, significantly promote microtubule more external Polymerization, and the activity of (3) number sample is higher than (2) number sample.
Claims (10)
1. method of from the slime bacteria fermented liquid, separating the purification ebormycine, this method concrete steps order is as follows:
(1) preparation of hybrid resin: with CD180, CAD-40, XDA, S-8, NKA-II, six kinds of resins of AB-8 mix by weight 0.8～1.2: 0.8～1.2: 1.8～2.2: 0.8～1.2: 2.8～3.2: 1.8～2.2, outstanding being dissolved in isopyknic distilled water is prepared into mixed resin solution;
(2) hybrid resin absorption: when carrying out the slime bacteria fermentation, the above-mentioned hybrid resin that adds 0.1%-5% (volume/volume) in liquid nutrient medium adsorbs, and sorbent material is present among the whole fermentation flow process, after the fermentation ends, filtration makes sedimentary polymeric adsorbent mixture, and is standby;
(3) solid-liquid stepwise solvent extraction: 1. use the methyl alcohol of 10-20 times of volume that the resin compound that step (2) obtains is extracted, be incubated 30-50 ℃, 24-48 hour, the centrifugal resin of removing obtained first part's extract; 2. with 40 ℃ of evaporated in vacuo of the methyl alcohol in first part's extract, pulverize extract, use the methylene dichloride of 10-20 times of volume to carry out the extraction of second step, be incubated 20-30 ℃, 24-48 hour, the centrifugal precipitation of removing, obtain the second section extract,, pulverize extract 3. with 30 ℃ of evaporated in vacuo of the methylene dichloride in the second section extract, use the normal hexane of 10-20 times of volume to carry out the extraction of the 3rd step, be incubated 20-30 ℃, 24-48 hour, centrifugal collecting precipitation, use the normal hexane flushing precipitation of 10% volume ratio, obtain the third part extract;
(4) sieve chromatography: above-mentioned third part extract heavily is dissolved in 2 times of volumes methanol, carries out dextrane gel (Sephadex LH-20) molecular sieve column chromatography with the application of sample amount of 2-5% column volume and separate; Moving phase is methyl alcohol or methylene dichloride, flow velocity be 0.5 column volume/hour, detect wavelength 254nm, the elution volume of ebormycine is a 1.5-2.5 times of column volume;
(5) crystallization: the collection specimen preparation of step (4) is become the supersaturated solution of ketone, utilize the crystallizer preparation and collect crystallization; Method is that thermostatic bath is heated to 45-60 ℃, sample is put into crystallizing dish, be incubated 15-30 minute, all dissolve to sample, begin gradient cooling then, speed is 0.5～3 ℃/30 minutes, after being cooled to 35-40 ℃, use solvent evaporated method instead, constant temperature concentrated liquid volume is to 1/10th of initial volume, can obtain a large amount of white powder crystallizations in the crystallizing dish bottom, be the mixture of ebomycin A and B;
(6) high performance liquid phase separates: if obtain the ebomycin A or the B of complete purifying, can adopt high performance liquid phase to carry out ebomycin A and B mixture separation, use anti-phase semipreparative column (Hypersil ODS2 C18), specification is 250 * 8mm, packing material size 10 μ m, flow velocity is 2ml/min, 249nm detects, 24 ℃ of column temperatures, sample size 100 μ l, detection time 30min, moving phase is 65% methyl alcohol and 35% water; The residence time of ebomycin A is 9.6 minutes, and the residence time of epothilone B is 11.1 minutes; Can obtain the ebomycin A or the B of purifying after the collection.
2. a kind of method of separating the purification ebormycine from the slime bacteria fermented liquid as claimed in claim 1 is characterized in that, the described CD180 of step (1), and CAD-40, XDA, S-8, NKA-II, the weight ratio of six kinds of mixed with resin of AB-8 is 1: 1: 2: 1: 3: 2.
3. a kind of method of separating the purification ebormycine from the slime bacteria fermented liquid as claimed in claim 1 is characterized in that described absorption with the hybrid resin addition of step (2) is the 0.5%-2% of liquid nutrient medium volume ratio.
4. a kind of method of separating the purification ebormycine from the slime bacteria fermented liquid as claimed in claim 1 is characterized in that, step (3) described 1. in, optimum extraction temperature is 35-40 ℃.
5. a kind of method of separating the purification ebormycine from the slime bacteria fermented liquid as claimed in claim 1 is characterized in that, step (3) described 2. in, optimum extraction temperature is 22-25 ℃.
6. a kind of method of separating the purification ebormycine from the slime bacteria fermented liquid as claimed in claim 1 is characterized in that, step (3) described 3. in, optimum extraction temperature is 25-28 ℃.
7. a kind of method of separating the purification ebormycine from the slime bacteria fermented liquid as claimed in claim 1 is characterized in that step (4) the suitableeest described application of sample amount is the 2.5-3.8% column volume.
8. a kind of method of separating the purification ebormycine from the slime bacteria fermented liquid as claimed in claim 1 is characterized in that, the elution volume of the described ebormycine of step (4), and when using methyl alcohol as moving phase, elution volume is a 1.8-2.3 times of column volume; When using methylene dichloride as moving phase, elution volume is a 1.5-2.1 times of column volume.
9. a kind of method of separating the purification ebormycine from the slime bacteria fermented liquid as claimed in claim 1 is characterized in that the supersaturated solution of the described ketone of step (5) is one or both in 2-butanone or the acetone.
10. a kind of method of separating the purification ebormycine from the slime bacteria fermented liquid as claimed in claim 1 is characterized in that the suitableeest speed of the described gradient cooling of step (5) is 0.5～1 ℃/30 minutes.
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|---|---|---|---|
| CNB021100675A CN1142163C (en) | 2002-02-07 | 2002-02-07 | Process for separating and purifying ebormycine from fermented myxobacterium liquid |
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| CNB021100675A CN1142163C (en) | 2002-02-07 | 2002-02-07 | Process for separating and purifying ebormycine from fermented myxobacterium liquid |
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| CN1369484A CN1369484A (en) | 2002-09-18 |
| CN1142163C true CN1142163C (en) | 2004-03-17 |
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| EP2280014B1 (en) * | 2002-09-23 | 2013-10-23 | Bristol-Myers Squibb Company | Methods for the preparation, isolation and purification of epothilone B |
| TWI409077B (en) * | 2006-04-18 | 2013-09-21 | Piramal Life Sciences Ltd | Novel antibacterial compounds |
| WO2009100571A1 (en) * | 2008-02-01 | 2009-08-20 | Zhejiang Hisun Pharmaceutical Co., Ltd. | A method for the separation and purification of epothilones |
| CN103145722B (en) * | 2013-03-05 | 2015-12-02 | 福建省微生物研究所 | A kind of method of high speed adverse current chromatogram separating-purifying ebormycine |
| CN103275098B (en) * | 2013-06-07 | 2015-07-08 | 江苏迪沃特仪器设备科技有限公司 | Method for separation purification of epothilone by using dynamic axial compression column |
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