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A method for producing paclitaxel by using yew seed endophyte

A technology of yew and endophytic bacteria, which is applied in the field of producing paclitaxel through the endophytic bacteria of yew seeds, and can solve the problem of a large gap in the paclitaxel raw material drug market

Inactive Publication Date: 2016-10-26
LIAOCHENG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problem that the paclitaxel crude drug market gap is relatively large, the purpose of the present invention is to provide a method for producing paclitaxel by yew seed endophytes

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] A method utilizing taxus seed endophyte to produce paclitaxel, comprising the following steps:

[0046] A) 'Mandia' yew fruit, remove the pulp, soak the skinned seeds with 2% detergent solution, shake the shaker at 100-150 for 15-20 minutes, then rinse with water for 15-30 minutes, and then The washed explants were sterilized with 75% alcohol for 30s, placed in 0.1% mercury chloride, soaked for sterilization for 9-11 minutes, rinsed with sterile deionized water for 6-8 times, and inoculated in the 0.3mg / L SH medium, including 30g / L sucrose, 7.5g / L agar powder, pH5.8;

[0047] B) After culturing for 3 days, take out the cultured yew seeds and sterilize them with 0.1% mercuric chloride for 10 minutes, and continue to culture them in SH medium containing 0.3 mg / L gibberellin, including 30 g / L sucrose and 7.5 g agar powder. g / L, pH5.8. The culture temperature is 21±2°C, the light intensity is 1600Lux, and the light time is 14 hours;

[0048] C) After 7 days of culture, i...

Embodiment 2

[0052] A method utilizing taxus seed endophyte to produce paclitaxel, comprising the following steps:

[0053] A) 'Mandia' yew fruit, remove the pulp, soak the seeds with 3% detergent solution, shake on the shaker for 15 minutes, rinse with tap water for 15 minutes, disinfect with 75% alcohol for 30 seconds, 0.1% mercuric chloride Sterilize for 9 minutes, place in SH medium containing 0.3 mg / L gibberellin, 30 g / L sucrose, 7.5 g / L agar powder, pH 5.8;

[0054] B) After culturing for 3 days, take out the cultured yew seeds and sterilize them for 10 minutes with 0.1% mercuric chloride, and culture them in hormone-free 0.3mg / L SH medium, including 30g / L sucrose and 7.5g agar powder / L, pH5.8.

[0055] C) After culturing for 8 days, isolate the fungi growing around the seeds, place them in PDA solid medium for cultivation, and then pick some plaques and place them in PDA liquid medium. The inoculum volume of each Erlenmeyer flask was 3%, the liquid volume was 200mL / 500mL, and c...

Embodiment 3

[0059] A method utilizing taxus seed endophyte to produce paclitaxel, comprising the following steps:

[0060] A) 'Mandia' yew fruit, remove the pulp, soak the seeds with 3% detergent solution, shake on the shaker for 15 minutes, rinse with tap water for 15 minutes, disinfect with 75% alcohol for 30 seconds, 0.1% mercuric chloride Sterilize for 9 minutes, place in SH medium containing 0.3 mg / L gibberellin, 30 g / L sucrose, 7.5 g / L agar powder, pH 5.8;

[0061]B) After culturing for 3 days, take out the cultured yew seeds and sterilize them for 10 minutes with 0.1% mercuric chloride, and culture them in hormone-free 0.3mg / L SH medium, including 30g / L sucrose and 7.5g agar powder / L, pH5.8.

[0062] C) After culturing for 8 days, isolate the fungi growing around the seeds, place them in PDA solid medium for cultivation, and then pick some plaques and place them in PDA liquid medium. The inoculum volume of each Erlenmeyer flask was 3%, the liquid volume was 200mL / 500mL, and cu...

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Abstract

The invention provides a method for producing taxol by utilizing Chinese yew seed endophyte. The method for producing the taxol by utilizing the Chinese yew seed endophyte comprises the following steps: sterilizing and inoculating while Chinese yew seeds are taken as explants, then carrying out secondary sterilization, culturing for a period of time, picking endophytic fungi at the periphery of the Chinese yew seeds, placing the endophytic fungi into a PDA solid culture medium, culturing, then picking part of bacterial plaque, placing the picked bacterial plaque into PDA liquid and culturing; filtering ferment with gauze, and extracting filtrate with ethyl acetate; carrying out reduced-pressure distillation at the temperature of 35 DEG C after filtering by adopting a rotary evaporator, and dissolving in a small amount of methyl alcohol, so that a methanol solution containing taxol is obtained. The method for producing the taxol by utilizing the Chinese yew seed endophyte has the advantages that the problems that Chinese yew plants grow slowly and chemical synthesis of the taxol is complex while a fungus culturing technique is applied can be solved, a foundation is provided for industrial production of the taxol by fungal fermentation, the method for producing the taxol by utilizing the Chinese yew seed endophyte is environment-friendly, raw materials are rich, and the problem of high supply shortage of the taxol can be well relieved.

Description

technical field [0001] The invention relates to the production of paclitaxel, in particular to a method for producing paclitaxel through taxus seed endophytes. Background technique [0002] Paclitaxel (Taxol), a diterpene compound isolated from Taxus spp., has a unique anti-cancer effect. It has high development and utilization value and is currently one of the mainstream anti-cancer drugs in the world. one. Chemical name: 5β,20-epoxy-1,2α,4,7β,10β,13α-hexahydroxytaxane-11-en-9-one-4,10-diacetate-2-benzoic acid Ester-13 [(2'R,3'S)-N-benzoyl-3-phenylisoserine ester]. The chemical formula is C 47 h 51 NO 14 , the molecular weight is 853.92, white crystalline powder, odorless, tasteless, insoluble in water, easily soluble in organic solvents such as chloroform and acetone, melting point 213~216℃. Paclitaxel was discovered in 1967 by Dr. Wall and Dr. Wani of the Triangle Research Institute in North Carolina, USA. Further studies have found that paclitaxel can tightly bind...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/02
Inventor 穆红梅
Owner LIAOCHENG UNIV
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