Method for producing paclitaxel by using calluses of blades of Taxus chinensis

A technology of callus and yew, applied in the field of paclitaxel production, to achieve the effect of increasing yield, rich raw materials, and fast growth of callus

Inactive Publication Date: 2014-09-17
LIAOCHENG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the relatively large gap in the paclitaxel raw material drug market, the purpose of the present invention is to provide a method for producing paclitaxel by yew leaf callus

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] A) Soak the leaves of Taxus 'Mandia' with 2% detergent solution, shake the shaker at 100-150 for 15 minutes, rinse with water for 15 minutes, and wash the explants with 75% alcohol Sterilize for 30s, place in mercuric chloride with a concentration of 0.1%, immerse and sterilize for 9 minutes, rinse with sterile deionized water for 8 times, inoculate in a solution containing 2,4-D 3.0mg / L, L-proline 300 mg / L SH medium, in which sucrose 30g / L, agar powder 7.5g / L, pH5.8;

[0039] B) After culturing for 5 days, inoculate the leaf callus of Taxus chinensis in SH+2,4-D1.0 mg / L + L-proline 300mg / L culture medium, the culture temperature is 21±2℃, light The intensity is 1200 Lux, the light time is 14 hours, and the culture time is 10 days.

[0040] C) After culturing for 10-15 days, paclitaxel content was detected, the callus was ground with liquid nitrogen, extracted with an equal volume of ethyl acetate, and the organic phase was collected by ultrasonication for 30 minutes...

Embodiment 2

[0044] A) Soak the leaves of Taxus 'Mandia' in 2% detergent solution, shake the shaker at 100-150 for 16 minutes, rinse with water for 18 minutes, and wash the explants with 75% alcohol Sterilize for 30s, place in mercuric chloride with a concentration of 0.1%, immerse and sterilize for 10 minutes, rinse with sterile deionized water for 8 times, inoculate in a solution containing 2,4-D 4.0mg / L, L-proline 400 mg / L SH medium, in which sucrose 30g / L, agar powder 7.5g / L, pH5.8;

[0045] B) After culturing for 5 days, inoculate the leaf callus of Taxus chinensis in SH+2,4-D3.0 mg / L + L-proline 300mg / L culture medium, the culture temperature is 21±2℃, light The intensity is 1300Lux, the light time is 14 hours, and the culture time is 10 days.

[0046] C) The paclitaxel content was detected after 13 days of culture, the callus was ground with liquid nitrogen, extracted with an equal volume of ethyl acetate, and the organic phase was collected by ultrasonication for 30 minutes. Th...

Embodiment 3

[0050] A) Soak the leaves of yew 'Mandia' in 2% detergent solution, shake the shaker at 130 rpm for 15 minutes, rinse with water for 20 minutes, and then disinfect the explants with 75% alcohol for 30 seconds , placed in mercuric chloride with a concentration of 0.1%, soaked and sterilized for 11 minutes, rinsed with sterile deionized water for 6 times, and inoculated in the In SH medium, sucrose 30g / L, agar powder 7.5g / L, pH 5.8;

[0051] B) After 7 days of culture, inoculate the leaf callus of Taxus chinensis in SH+2,4-D1.0 mg / L + L-proline 300mg / L culture medium, the culture temperature is 21±2℃, light The intensity is 1300Lux, the light time is 12 hours, and the culture time is 15 days.

[0052] C) The paclitaxel content was detected after 15 days of culture, the callus was ground with liquid nitrogen, extracted with an equal volume of ethyl acetate, and the organic phase was collected by ultrasonication for 30 min. The aqueous phase was extracted once with an equal vol...

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PUM

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Abstract

The invention provides a method for producing paclitaxel by using calluses of blades of Taxus chinensis. The method comprises the following steps: by taking blades of taxus madia as explants, disinfecting, sterilizing, inoculating and culturing for a period of time; generating calluses at the wounds of the blades and continuously carrying out multiplication culture on the calluses; grinding the calluses of blades of Taxus chinensis and carrying out ultrasonic extraction for 3 times by ethyl acetate; collecting an organic phase; filtering and carrying out vacuum distillation by a rotary evaporator; and dissolving in a small amount of methanol to obtain methanol solution containing paclitaxel. The method provided by the invention overcomes the problem that the Taxus chinensis plant is slow in growing and chemical synthesis of paclitaxel is complex by adopting the tissue culture technique. The minimal medium used is based on SH (Schenk and Hildebrand) medium and supplemented with components such as 2, 4-dichlorphenoxyacetic acid, L-proline and saccharose. The method provides a base to industrialized cell production of paclitaxel, is environmentally friendly and abundant in raw material and can well alleviate the problem on supply gap of paclitaxel in the market.

Description

technical field [0001] The present invention relates to the production of paclitaxel, in particular to a method for producing paclitaxel through yew leaf callus. Background technique [0002] Taxol, chemical formula C47H51NO14, molecular weight 853.92, white crystalline powder, odorless, tasteless, insoluble in water, soluble in chloroform, acetone and other organic solvents, melting point 213-216 ℃. 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]. It is a diterpene compound isolated from Taxus spp. with unique anti-cancer effect. It has high development and utilization value and is officially approved by the US National Center for Food and Drug Administration (1992.12) , one of the world's mainstream anticancer drugs. Initially, paclitaxel was a drug for the treatment of advanced ovarian cancer. It was subsequently approved for the treatment of breast cancer....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
Inventor 穆红梅杜秀菊曹贵玲
Owner LIAOCHENG UNIV
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