CN1369484A - Process for separating and purifying ebormycine from fermented myxobacterium liquid - Google Patents

Abstract

A process for extracting Epophilone from fermented liquid of myxobacterium includes mixed resin adsorption, solid and liquid extractions, molecular sieve chromatography, crystallizing and effiicent liquid-phase separation. Its advantages are high purity and extraction rate (more than 80%). The said Epophilone is an antineoplastic compound.

Description

A method for separating and purifying epothilone from myxobacteria fermentation broth

(1) Technical field

The invention relates to a method for separating and purifying macrolide compounds from myxobacteria fermentation liquid, in particular to a method for separating and purifying epothilone from myxobacteria fermentation liquid.

(2) Background technology

At present, the most clinically successful anti-tumor chemotherapy drug is the microtubule polymerization-promoting natural compound paclitaxel (Taxol ^® ) and its analogue taxotere (docetaxel, Taxotere ^® ), which are used for ovarian cancer, thymus cancer, colon cancer, etc. Treatment of solid cancers such as cancer, lung cancer and liver cancer. The success of the inducible anti-tumor drug paclitaxel and its shortcomings in chemotherapy (low water solubility and cell drug resistance during chemotherapy, etc.) have prompted researchers to further screen for drugs with better chemical properties, biological properties and pharmacology. properties of microtubule stabilizers. However, after a long time and a lot of screening, it was not until recent years that four new types of natural compounds were discovered - eleutherobin, discodermolide, laulimalides and epothilone (Epothilone) has a microtubule stabilizing function. Wherein, in 1993, Heffler (Hfle) etc. reported that new 16-membered macrolide compounds Epothilone (Epothilone) A and B were isolated from myxobacterium cellulosum (Sorangium cellulosum). In 1995, Bollag et al. discovered the microtubule polymerization-promoting activity of epothilone in the large-scale screening of anti-tumor compounds, which aroused widespread concern. Like paclitaxel, epothilone induces tubulin to form microtubules at low temperature and in the absence of guanosine triphosphate (GTP) or microtubule-associated proteins (MAPs). Unlike paclitaxel, epothilones remain active against p-glycoprotein-expressing multidrug-resistant (MDR) cell lines and tumors. In paclitaxel-sensitive cell lines, epothilone B has greater inhibitory activity than epothilone A or paclitaxel. On the other hand, the endotoxin-like activity of paclitaxel may cause non-hematological side effects in clinical chemotherapy, while epothilone does not trigger the endotoxin signaling pathway of cells. In addition, studies by Taraschi et al. have shown that microtubule stabilizers such as paclitaxel or epothilone A also have antimalarial activity and can hinder the formation of merozoites.

Compared with paclitaxel, epothilone has a simpler structure, good water solubility and great medicinal potential, so that people have invested great enthusiasm in research, in order to develop it into an anti-tumor drug more quickly. Chemists have put a lot of energy into the synthesis and separation of epothilones and their analogues, but for the separation and purification of epothilones from fermentation broth, the methods of Gerth et al. have been used at present. The method comprises four steps of resin adsorption, silica gel column gradient separation, molecular sieve chromatography and high performance liquid phase separation. Our research shows that this method has certain defects and deficiencies, especially that it cannot meet various requirements of industrial production. Mainly manifested in: the steps are too complicated, so that the efficiency of the operation is low and the process cost is very high; the yield of the product is low, no more than 30%, and the process loss is as high as 70%; during resin adsorption, use Amberlite (Amberlite) XAD -16 resin, the cost is very high, and there is no resin substitute of the same model in China at present; To make epothilone A and B reach the drug purity (greater than 90%), it must be separated through high-efficiency liquid phase, which is in industrial production is difficult to achieve. After searching the literature and patents, there is no major progress and related patents in this direction at home and abroad.

(3) Contents of the invention

In view of the above defects, the main purpose of the present invention is to provide a method for separating and purifying epothilones from myxobacteria fermentation broth. The method can effectively overcome the disadvantages of the existing process, achieve the purpose of efficiently and economically separating and purifying the epothilone from the myxobacteria fermentation broth, thereby realizing the industrialization of the extraction process.

The object of the present invention is achieved through the following technical solutions, and the sequence of concrete steps is as follows:

(1) Preparation of mixed resin: CD180, CAD-40, XDA, S-8, NKA-II, AB-8 six resins in weight ratio 0.8～1.2: 0.8～1.2: 1.8～2.2: 0.8～1.2: Mix 2.8～3.2: 1.8～2.2, suspend and dissolve in an equal volume of distilled water, and prepare a mixed resin solution;

(2) Mixed resin adsorption: when carrying out myxobacteria fermentation, add 0.1%-5% (volume/volume) of the above-mentioned mixed resin to the liquid medium for adsorption, and the adsorbent exists in the whole fermentation process. , filtered to obtain the precipitated adsorption resin mixture, for subsequent use;

(3) Solid-liquid step-by-step extraction: ①Use 10-20 times the volume of methanol to extract the resin mixture obtained in step (2), keep it warm at 30-50°C for 24-48 hours, centrifuge to remove the resin, and obtain the first part of the extract ;② Evaporate the methanol in the first part of the extract to dryness at 40°C in vacuum, crush the extract, use 10-20 times the volume of dichloromethane for the second step of extraction, keep warm at 20-30°C for 24-48 hours, and centrifuge to remove the precipitate , to obtain the second part of the extract, ③ evaporate the dichloromethane in the second part of the extract to dryness under vacuum at 30°C, pulverize the extract, use 10-20 times the volume of n-hexane to carry out the third step of extraction, and keep warm at 20-30°C , 24-48 hours, centrifuge to collect the precipitate, use 10% volume ratio of n-hexane to wash the precipitate, and obtain the third part of the extract;

(4) Molecular sieve chromatography: redissolve the third part of the above extract in 2 times the volume of methanol, and perform Sephadex LH-20 molecular sieve column chromatography with a sample volume of 2-5% column bed volume Separation; the mobile phase is methanol or dichloromethane, the flow rate is 0.5 column bed volume/hour, the detection wavelength is 254nm, and the elution volume of epothilone is 1.5-2.5 times the column volume;

(5) Crystallization: Prepare the collected samples in step (4) into a supersaturated solution of ketones, and use a crystallizer to prepare and collect crystals; the method is to heat the constant temperature tank to 45-60°C, put the sample into a crystallization dish, and keep it warm 15-30 minutes, until all the samples are dissolved, then start gradient cooling at a rate of 0.5-3°C/30 minutes, after cooling down to 35-40°C, use solvent evaporation method to concentrate the liquid volume at constant temperature to one tenth of the original volume 1. A large amount of white powdery crystals can be obtained at the bottom of the crystallization dish, which is a mixture of epothilone A and B;

(6) High-efficiency liquid phase separation: If completely purified epothilone A or B is obtained, high-efficiency liquid phase can be used to separate the mixture of epothilone A and B, using a reversed-phase semi-preparative column (Hypersil ODS2 C18), specifications 250×8mm, filler particle size 10μm, flow rate 2ml/min, detection at 249nm, column temperature 24°C, injection volume 100μl, detection time 30min, mobile phase 65% methanol and 35% water; Epothilone A The retention time was 9.6 minutes and that of epothilone B was 11.1 minutes; purified epothilone A or B was obtained after collection.

Wherein the weight ratio of CD180, CAD-40, XDA, S-8, NKA-II, AB-8 six kinds of resins described in the step (1) is 1:1:2:1:3:2.

Wherein the amount of the mixed resin for adsorption described in step (2) is 0.5%-2% of the volume ratio of the liquid medium.

Wherein step (3) described in ①, the most suitable extraction temperature is 35-40 ℃.

Wherein step (3) described in ②, the most suitable extraction temperature is 22-25 ℃.

Wherein step (3) described in ③, the most suitable extraction temperature is 25-28 ℃.

Wherein the optimum sample amount described in step (4) is 2.5-3.8% column bed volume.

Wherein the elution volume of epothilone described in step (4), when using methanol as the mobile phase, the elution volume is 1.8-2.3 times the column volume; when using dichloromethane as the mobile phase, the elution volume is 1.5-2.1 times the column volume.

Wherein the ketone supersaturated solution described in step (5) is one or both of 2-butanone or acetone.

Wherein the optimum rate of gradient cooling described in step (5) is 0.5-1° C./30 minutes.

For the epothilone A and B prepared by separation and purification, respectively measure mass spectrum (see accompanying drawing 4), ultraviolet spectrogram (see accompanying drawing 5), infrared spectrogram (see accompanying drawing 6) and nuclear magnetic resonance spectrogram (see accompanying drawing 6) See accompanying drawing 7 and accompanying drawing 8), confirm the structure (see accompanying drawing 3). Also, their antitumor activity (see Appendix 1) and microtubule polymerization promoting activity (see Appendix 2) were determined.

In the method of the present invention, the adsorption rate of the mixed resin for epothilones is 95-100%, the extraction rate of epothilones is 85-90% when the crystallization step is completed, and the purity is 95-97%. After phase separation, the extraction rate of epothilone A and B is 82-89%, and the purity is 99.9-99.99%.

The creativity of the present invention lies in that it has developed a low-cost, high-efficiency mixed adsorption resin, whose adsorption rate is as high as 95-100%, replacing the expensive imported adsorption resin; developed a step-by-step solid-liquid extraction technology to replace the silica gel column in the original step Gradient elution separation greatly improves the final extraction yield and reduces the cost; develops the crystallization process, and develops a special crystallizer, and creatively purifies epothilone through crystallization, so that when the crystallization step is completed, epothilone The extraction rate of the mycins A and B is 85-90%, and the purity is as high as 95-97%, which fully meets the requirements for preparing medicines. However, in the original extraction process, the purity of epothilone was less than 70% before high-performance liquid phase separation, so it could not be directly prepared as a drug. The main advantage of the present invention is that the extraction yield of the anti-tumor compound epothilone can reach more than 80% through processes such as step-by-step solid-liquid extraction and crystallization, and the cost is greatly reduced due to the use of cheap mixed resins for adsorption. Increased efficiency. Considering that in industrial production, epothilone A and B have roughly the same drug efficacy and toxicity, and can be mixed for pharmaceuticals, so the product can be formed after completing the crystallization part, which greatly improves the feasibility and scalability of industrial production. This invention has created an innovative technology for the efficient and economical separation and purification of the anti-tumor compound epothilone. It has the feasibility and scalability of industrial production, and has good economic benefits and application prospects.

(4) Description of drawings Fig. 1. Process flow chart of separating and purifying epothilone from myxobacteria fermentation broth

Among them: 1. Mixed resin configuration; 2. Mixed resin adsorption; 3. Solid-liquid fractional extraction; 4. Molecular sieve chromatography; 5. Crystallization; 6. High performance liquid phase separation; Mixed crystallization of epothilone A and B. Figure 2. Schematic diagram of crystallizer structure for crystallization operation

Among them: 9 sample crystallization dish; 10 thermometer; 11 sample tank; 12 sample tank sealing cover; 13 single-phase synchronous motor; 14 electric stirrer controller; 15 electric coupling transformer; ; 19 heating device; 20 temperature controller; 21 double distilled water; 22 micro vacuum pump; 23 vacuum exhaust pipe. Figure 3. Chemical structures of Epothilone A and B

As shown in the figure, the R of epothilone A is H; the R of epothilone B is CH ₃ . Figure 4. Mass spectra of purified epothilone A and B

As shown in the figure, the sample in (4-A) is purified epothilone A, the (M+H) ⁺ of the component is 494.2561, and the molecular formula of the fitted compound is C ₂₆ H ₃₉ O ₆ NS , the relative deviation is 1.922e-06; the sample in (4-B) is purified epothilone B, the (M+H) ⁺ of the component is 508.2725, and the molecular formula of the fitted compound is C ₂₇ H ₄₁ O ₆ NS, with a relative deviation of 4.942e-07, are in perfect agreement with the theoretical values of epothilone A or B. Figure 5. UV spectra of purified epothilone A and B

As shown in the figure, the purified epothilones A and B have the same ultraviolet spectrum, and the wavelength values and molar extinction coefficients of the characteristic peaks are λ _max1 = 210, logε ₁ = 4.17, λ _max2 = 249, logε ₂ =3.97, completely consistent with the theoretical value of epothilone A or B. Figure 6. The infrared spectrum of the purified epothilone B

As shown in the figure, the infrared spectrogram of the purified epothilone B completely agrees with the theoretical value of epothilone B. Figure 7. PMR spectra of purified epothilone A and B

As shown in the figure, the sample of (7-A) figure is the purified epothilone A; the sample of (7-B) figure is the purified epothilone B, and the sample solution is deuterated methanol, and the result It is in perfect agreement with the theoretical value of epothilone A or B. Figure 8. ¹³ C-NMR spectra of purified epothilone A and B

As shown in the figure, the sample of (8-A) figure is the purified epothilone A; the sample of (8-B) figure is the purified epothilone B, and the sample solution is deuterated methanol, and the result It is in perfect agreement with the theoretical value of epothilone A or B.

Figure 9. Diagram of detection results by variable temperature ultraviolet spectrophotometry. Appendix 1: In vitro anti-tumor activity test report of purified epothilone A and B

The conclusion shows that: when the concentration of purified epothilone A is greater than 50ng/ml, two kinds of tumor cells can be killed; when the concentration of purified epothilone B is greater than 10ng/ml, two kinds of tumor cells can be killed . Both samples have obvious in vitro antitumor activity, which is consistent with the literature value. Attachment 2: Experiment report on microtubule polymerization-promoting activity of purified epothilone A and B

The conclusion shows that both the purified epothilones A and B can significantly promote the polymerization of microtubules in vitro in the absence of GTP, and the activity of B is higher than that of A, which is consistent with the conclusions in the literature.

(5) Specific embodiments Example 1: The present invention utilizes technical means such as mixed resin adsorption, solid-liquid extraction, molecular sieve chromatography, crystallization and high-efficiency liquid phase separation to separate from a kind of myxobacterium S. cellulosus fermentation broth Extract epothilone, the sequence of concrete steps is as follows: (1) preparation of mixed resin: CD180, CAD-40, XDA, S-8, NKA-II, six kinds of resins of AB-8 are weighed

The volume ratio is 1:1:2:1:3:2, suspended and dissolved in distilled water of equal volume to prepare a mixed resin solution. (2) Mixed resin adsorption: when carrying out the fermentation of S. cellulosus, add 2% (volume

Volume/volume) of mixed resin for adsorption, the adsorbent exists in the whole fermentation process, after the fermentation,

The obtained precipitated adsorption resin mixture was filtered and set aside. (3) Solid-liquid step-by-step extraction:

①Use 15 times the volume of methanol to extract the resin mixture obtained in step (2), keep it warm at 38°C for 36 hours, centrifuge to remove the resin, and obtain the first part of the extract;

② Evaporate the methanol in the first part of the extract to dryness under vacuum at 40°C, pulverize the extract, use 15 times the volume of dichloromethane for the second step of extraction, keep warm at 24°C for 36 hours, centrifuge to remove the precipitate, and obtain the second part of the extract ;

③ Evaporate the dichloromethane in the second part of the extract to dryness under vacuum at 30°C, crush the extract, use 15 times the volume of n-hexane for the third step of extraction, keep warm at 27°C for 36 hours, collect the precipitate by centrifugation, and use 10% volume Rinse and precipitate with normal hexane to obtain the third part of the extract; (4) molecular sieve chromatography: the above-mentioned third part of the extract was redissolved in 2 times the volume of methanol, and the dextran was carried out with a sample amount of 3% column bed volume. Gel (Sephadex LH-20) molecular sieve column chromatography separation; the mobile phase is methanol, the flow rate is 0.5 column bed volume/hour, the detection wavelength is 254nm, and the elution volume of epothilone is 2.1 times the column volume; (5) crystallization : the collected sample of step (4) is prepared into a supersaturated solution of 2-butanone, and crystallization is prepared and collected using a crystallizer (see accompanying drawing 2 for the structure of the crystallizer); the method is to heat the thermostat to 50° C. Put it into a crystallization dish, keep it warm for 25 minutes until the sample is completely dissolved, then start the gradient cooling at a rate of 0.8°C/30 minutes, after cooling down to 38°C, use the solvent evaporation method to concentrate the liquid volume at a constant temperature to one tenth of the original volume One, a large amount of white powdery crystals can be obtained at the bottom of the crystallization dish, which is a mixture of epothilone A and B; (6) high-efficiency liquid phase separation: if completely purified epothilone A or B is obtained, use The mixture of epothilone A and B was separated by high performance liquid phase, using a reversed-phase semi-preparative column (Hypersil ODS2 C18), the specification is 250×8mm, the particle size of the filler is 10μm, the flow rate is 2ml/min, the detection is at 249nm, and the column temperature is 24°C , the injection volume is 100 μl, the detection time is 30min, the mobile phase is 65% methanol and 35% water; the retention time of epothilone A is 9.6 minutes, and the retention time of epothilone B is 11.1 minutes; it can be obtained after collection Purified epothilone A or B.

In the method of the present invention, the adsorption rate of the mixed resin for epothilones is 100%, and when the crystallization step is completed, the extraction rate of epothilones is 90%, and the purity is 97%. The extraction rate of bleomycin A and B is 89%, and the purity is 99.99%. Embodiment 2: The present invention utilizes technical means such as mixed resin adsorption, solid-liquid fractional extraction, molecular sieve chromatography, crystallization and high-efficiency liquid phase separation, separates and extracts epothilone from a kind of myxobacterium S. cellulosus fermentation broth, Concrete step sequence is as follows: (1) the preparation of mixed resin: CD180, CAD-40, XDA, S-8, NKA-II, AB-8 six kinds of resins are by weight 0.8: 0.8: 1.8: 0.8: 2.8: 1.8 Mix, suspend and dissolve in an equal volume of distilled water to prepare a mixed resin solution. (2) Mixed resin adsorption: when carrying out S. cellulosus fermentation, add 1% (volume/volume) mixed resin to 1000ml liquid culture medium and carry out adsorption, adsorbent exists in the whole fermentation process, after fermentation finishes, The obtained precipitated adsorption resin mixture was filtered and set aside. (3) Solid-liquid step-by-step extraction:

① Use 10 times the volume of methanol to extract the resin mixture obtained in step (2), keep it warm at 31°C for 46 hours, centrifuge to remove the resin, and obtain the first part of the extract;

② Evaporate the methanol in the first part of the extract to dryness under vacuum at 40°C, pulverize the extract, use 10 times the volume of dichloromethane for the second step of extraction, keep warm at 20°C for 46 hours, centrifuge to remove the precipitate, and obtain the second part of the extract ;

③ Evaporate the dichloromethane in the second part of the extract to dryness in vacuo at 30°C, crush the extract, use 10 times the volume of n-hexane for the third step of extraction, keep warm at 21°C for 46 hours, collect the precipitate by centrifugation, and use 10% volume Rinse and precipitate with normal hexane to obtain the third part of the extract; (4) Molecular sieve chromatography: the above-mentioned third part of the extract was redissolved in 2 times the volume of methanol, and the dextran was carried out with a sample amount of 2% column bed volume Gel (Sephadex LH-20) molecular sieve column chromatography separation; mobile phase is dichloromethane, flow velocity is 0.5 column bed volume/hour, detection wavelength 254nm, the elution volume of epothilone is 1.9 times column volume; (5 ) crystallization: the collected sample in step (4) is prepared into a supersaturated solution of acetone, and the crystallizer is used to prepare and collect the crystallization (see accompanying drawing 2 for the structure of the crystallizer); the method is to heat the thermostat to 45° C. In the crystallization dish, keep warm for 30 minutes until the sample is completely dissolved, and then start the gradient cooling at a rate of 0.5°C/30 minutes. After cooling down to 36°C, use the solvent evaporation method to concentrate the liquid volume at a constant temperature to one tenth of the original volume. , a large amount of white powdery crystals can be obtained at the bottom of the crystallization dish, which is a mixture of epothilone A and B; (6) high-efficiency liquid phase separation: if completely purified epothilone A or B is obtained, high-efficiency The mixture of epothilone A and B was separated in liquid phase, using a reversed-phase semi-preparative column (Hypersil ODS2 C18), with a specification of 250×8mm, a packing particle size of 10μm, a flow rate of 2ml/min, 249nm detection, and a column temperature of 24°C. The injection volume is 100 μl, the detection time is 30 minutes, the mobile phase is 65% methanol and 35% water; the retention time of epothilone A is 9.6 minutes, and the retention time of epothilone B is 11.1 minutes; after collection, the purified Epothilone A or B.

In the method of the present invention, the adsorption rate of the mixed resin for epothilones is 95%, and when the crystallization step is completed, the extraction rate of epothilones is 90%, and the purity is 95%. The extraction rate of bleomycin A and B is 82%, and the purity is 99.91%. Embodiment 3: The present invention utilizes technical means such as mixed resin adsorption, solid-liquid stepwise extraction, molecular sieve chromatography, crystallization and high-efficiency liquid phase separation, separates and extracts epothilone from a kind of myxobacterium S. cellulosus fermentation broth, Concrete step sequence is as follows: (1) the preparation of mixed resin: CD180, CAD-40, XDA, S-8, NKA-II, AB-8 six kinds of resins are by weight 1.2: 1.2: 2.3: 1.2: 3.2: 2.3 Mix, suspend and dissolve in an equal volume of distilled water to prepare a mixed resin solution. (2) Mixed resin adsorption: when carrying out S. cellulosus fermentation, add 1% (volume/volume) mixed resin to 5000ml liquid culture medium and carry out adsorption, adsorbent exists in the whole fermentation process, after fermentation finishes, The obtained precipitated adsorption resin mixture was filtered and set aside. (3) Solid-liquid step-by-step extraction:

①Use 20 times the volume of methanol to extract the resin mixture obtained in step (2), keep it warm at 50°C for 28 hours, centrifuge to remove the resin, and obtain the first part of the extract;

② Evaporate the methanol in the first part of the extract to dryness in vacuo at 40°C, crush the extract, use 20 times the volume of dichloromethane for the second step of extraction, keep warm at 30°C for 26 hours, centrifuge to remove the precipitate, and obtain the second part of the extract ;

③ Evaporate the dichloromethane in the second part of the extract to dryness in vacuo at 30°C, crush the extract, use 20 times the volume of n-hexane for the third step of extraction, keep warm at 30°C for 28 hours, collect the precipitate by centrifugation, and use 10% volume Rinse and precipitate with normal hexane to obtain the third part of the extract; (4) Molecular sieve chromatography: the above-mentioned third part of the extract was redissolved in 2 times the volume of methanol, and the dextran was carried out with a sample amount of 5% column bed volume Gel (Sephadex LH-20) molecular sieve column chromatography separation; mobile phase is dichloromethane, flow velocity is 0.5 column bed volume/hour, detection wavelength 254nm, the elution volume of epothilone is 2.1 times of column volume; (5 ) crystallization: the collected sample of step (4) is prepared into a supersaturated solution mixed with equal volumes of acetone and 2-butanone, and crystallization is prepared and collected using a crystallizer (see accompanying drawing 2 for the structure of the crystallizer); the method is to heat the thermostat to 60°C, put the sample into a crystallization dish, keep it warm for 15 minutes, until the sample is completely dissolved, then start gradient cooling at a rate of 1.5°C/30 minutes, after cooling down to 40°C, use the solvent evaporation method to concentrate the liquid volume at a constant temperature To one tenth of the original volume, a large amount of white powdery crystals can be obtained at the bottom of the crystallization dish, which is the mixture of epothilone A and B; (6) high-efficiency liquid phase separation: if the completely purified epothilone Element A or B, the mixture of epothilone A and B can be separated by high performance liquid phase, using a reversed-phase semi-preparative column (Hypersil ODS2 C18), the specification is 250 × 8mm, the particle size of the filler is 10 μm, and the flow rate is 2ml/min. 249nm detection, column temperature 24°C, injection volume 100μl, detection time 30min, mobile phase 65% methanol and 35% water; retention time of epothilone A is 9.6 minutes, retention time of epothilone B is 11.1 Minutes; purified epothilone A or B can be obtained after collection.

In the method of the present invention, the adsorption rate of the mixed resin for epothilones is 99.5%, and when the crystallization step is completed, the extraction rate of epothilones is 90%, and the purity is 97%. The extraction rate of bleomycin A and B is 89%, and the purity is 99.97%. Appendix 1: In vitro anti-tumor activity test report of purified epothilone A and B

Test report Sample name: Purified Epothilone A and B Submitter: State Key Laboratory of Biotechnology, School of Life Sciences, Shandong University Submitter: Li Yuezhong Test purpose: To test whether the two samples can kill Date of receipt of dead tumor cells Hela and Bel-7402: 2001.5.29 Date of report: 2001.6.8 Samples: (1) buffer control sample; (2) epothilone A; (3) epothilone B. Detection method: MTT method The test results are reported as follows: Test cell line: Hela sample concentration 1000ng/ml 100ng/ml 50ng/ml 10ng/ml 5ng/ml 1ng/ml sample(1) - - - - - - Sample(2) + + + - - - Sample(3) + + + + + - Test cell line: Bel-7402 sample concentration 1000ng/ml 100ng/ml 50ng/ml 10ng/ml 5ng/ml 1ng/ml sample(1) - - - - - - Sample(2) + + + - - - Sample(3) + + + + - - Conclusion: (2) sample can kill two tumor cells when the concentration is greater than 50ng/ml; (3) sample can kill two tumor cells when the concentration is greater than 10ng/ml. Both samples have obvious antitumor activity in vitro. Attachment 2: Experiment report on microtubule polymerization-promoting activity of purified epothilone A and B

Test report Sample name: Purified Epothilone A and B Submitter: State Key Laboratory of Biotechnology, School of Life Sciences, Shandong University Submitter: Li Yuezhong Test purpose: To test whether the two samples can Conditions that promote in vitro polymerization of microtubules Received date: 2001.6.2 Reported date: 2001.6.16 Samples: (1) buffer control sample; (2) epothilone A; (3) epothilone B. Detection method: The temperature-variable ultraviolet spectrophotometry detection result report is shown in Figure 9. Conclusion: Both samples (2) and (3) can significantly promote the polymerization of microtubules in vitro in the absence of GTP, and the activity of sample (3) is higher than that of sample (2).

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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