Method for preparing epothilones B
An epothilone and adsorbent technology, applied in the field of chronic myeloid leukemia drugs, can solve the problems of complex drug resistance mechanism, and achieve the effects of easy large-scale production, strong applicability and simple operation
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Embodiment 1
[0037] Embodiment one Seed liquid culture
[0038] Take the frozen strains and transfer them to 50ml medium, the medium components are potato starch 8g / L, glucose 2g / L, soybean powder 2g / L, yeast extract 2g / L, etc. After culturing for 24 hours, transfer to a plurality of 500ml No. I culture medium, cultivate the seed solution for 72 hours, and transfer to a fermenter for fermentation.
Embodiment 2
[0039] Embodiment two fermentation, extraction crude extract
[0040] 1) Add 65L medium to a 100L fermenter, and the medium components are potato starch 1g / L, glucose 10g / L, soybean powder 30g / L, yeast extract 15g / L, etc. Sterilize at 121°C for 20 minutes. Transplant under aseptic conditions. During the fermentation process, set the ventilation rate to 25L / min, the pressure to 0.4Mpa, the rotational speed to 100r / min, the temperature to 32°C, and the pH to 7.4. After culturing for 12 hours, 1% of the total volume of AB-8 macroporous adsorption resin was added. Harvest after culturing for 100 h.
[0041] Filter the fermented liquid with a thick cloth to obtain the adsorbent AB-8, and extract it twice with 2 times the volume of isopropanol for 1 hour each time. The isopropanol solutions were combined, concentrated to 1 / 10 by distillation under reduced pressure, and the concentrated isopropanol was extracted three times with ethyl acetate to obtain a crude epothilone B extract...
Embodiment 3
[0048] Embodiment three: Separation and purification of epothilone B
[0049] The crude extract containing epothilone B is separated and purified by two steps to obtain epothilone B with a purity higher than 95%.
[0050] The first step: Sephadex LH-20 column chromatography
[0051] After the extract was dissolved in methanol, the supernatant was collected by centrifugation and added to the top of Sephadex LH-20 column. Mobile phase: methanol, the mobile phase flow rate is 10cm / h. Analysis by HPLC high performance liquid chromatography (filler: Kromasil C 18 5μm, size: 4.6mm×150mm. Mobile phase: methanol: water = 7:3. Detection wavelength: UV230nm). Fractions containing epothilone B were combined for further purification.
[0052] The second step: C18 reversed-phase chromatographic separation
[0053] The fraction separated by Sephadex LH-20 was further purified by C18 reverse phase chromatography column. Mobile phase: methanol: water = 70:30, detection wavelength: UV2...
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