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Method for producing paclitaxel and its precursor baccatin iii by using suspension culture of non-embryogenic cells of Torreya hainanensis

A cell suspension, non-embryogenic technology, applied in the direction of fermentation, etc., to achieve the effect of rapid cell division, great potential for development and utilization, and stable yield

Inactive Publication Date: 2016-04-20
TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no cell engineering method to produce paclitaxel and its precursor baccatin III from suspension culture of Torreya hainanensis

Method used

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  • Method for producing paclitaxel and its precursor baccatin iii by using suspension culture of non-embryogenic cells of Torreya hainanensis
  • Method for producing paclitaxel and its precursor baccatin iii by using suspension culture of non-embryogenic cells of Torreya hainanensis
  • Method for producing paclitaxel and its precursor baccatin iii by using suspension culture of non-embryogenic cells of Torreya hainanensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Obtaining the Suspension Line of Non-embryogenic Cells of Torreya hainanensis and Determination of Paclitaxel Content

[0057] (1) Acquisition of non-embryogenic cell suspension line of Torreya hainanensis

[0058] (1) Take the annual leaves of Hainan rough torreya, the length of the leaves is 1-2cm, after the surface is disinfected, cut them into 1cm sections, and inoculate them on the callus induction medium [(NH 4 ) 2 SO 4 134mg / L+H 3 BO 3 3mg / L+CaCl 2 113.24mg / L+CoCl 2 ·6H 2 O0.025mg / L+CuSO 4 ·5H 2 O0.025mg / L+Na 2 EDTA·2H 2 O37.26mg / L+MgSO 4 122.09mg / L+MnSO 4 ·H 2 O10mg / L+Na 2 MoO 4 2H 2 O0.25mg / L+KI0.75mg / L+KNO 3 2500mg / L+NaH 2 PO 4 150mg / L+ZnSO 4 ·7H 2 O2mg / L+inositol 100mg / L+niacin 1mg / L+pyridoxine hydrochloride 1mg / L+thiamine hydrochloride 10mg / L+6-BA0.1mg / L+NAA1mg / L+2,4-D1mg / L+sucrose 30g / L +1.0% agar, pH is 5.85], dark culture, culture temperature is 28 ℃, obtain non-embryogenic callus after 28 days of culture, and this callus grows along...

Embodiment 2

[0067] The extraction process of paclitaxel and its precursor baccatin Ⅲ from the non-embryogenic cell suspension line of Torreya hainanensis is as follows:

[0068] (1) Acquisition of non-embryogenic cell suspension line of Torreya hainanensis

[0069] (1) Take Hainan rough torreya torreya leaves in the current year, the length of the leaves is 1-2cm, after the surface is disinfected and treated, cross-cut them into two parts, and inoculate them on the callus induction medium [(NH 4 ) 2 SO 4 132mg / L+H 3 BO 3 2.8mg / L+CaCl 2 112mg / L+CoCl 2 ·6H 2 O0.023mg / L+CuSO 4 ·5H 2 O0.022mg / L+Na 2 EDTA·2H 2 O36.8mg / L+MgSO 4 121.8mg / L+MnSO 4 ·H 2 O9mg / L+Na 2 MoO 4 2H 2 O0.24mg / L+KI0.65mg / L+KNO 3 2400mg / L+NaH 2 PO 4 140mg / L+ZnSO 4 ·7H 2 O1.8mg / L+inositol 90mg / L+niacin 0.9mg / L+pyridoxine hydrochloride 0.9mg / L+thiamine hydrochloride 9mg / L+6-BA0.09mg / L+NAA0.09mg / L+2,4-D0 .09mg / L+sucrose 20g / L+1.0% agar, pH is 5.6, dark culture, culture temperature is 28 ℃, obtain non-embryo...

Embodiment 3

[0077] The extraction process of paclitaxel and its precursor baccatin Ⅲ from the non-embryogenic cell suspension line of Torreya hainanensis is as follows:

[0078] (1) Acquisition of non-embryogenic cell suspension line of Torreya hainanensis

[0079] (1) Take Hainan rough torreya torreya leaves in the current year, the length of the leaves is 1-2cm, after the surface is disinfected and treated, cross-cut them into two parts, and inoculate them on the callus induction medium [(NH 4 ) 2 SO 4 136mg / L+H 3 BO 3 3.2mg / L+CaCl 2 114mg / L+CoCl 2 ·6H 2 O0.027mg / L+CuSO 4 ·5H 2 O0.028mg / L+Na 2 EDTA·2H 2 O37.6mg / L+MgSO 4 122.4mg / L+MnSO 4 ·H 2 O11mg / L+Na 2 MoO 4 2H 2 O0.26mg / L+KI0.85mg / L+KNO 3 2600mg / L+NaH 2 PO 4 160mg / L+ZnSO 4 ·7H 2 O2.2mg / L+inositol 110mg / L+niacin 1.1mg / L+pyridoxine hydrochloride 1.1mg / L+thiamine hydrochloride 11mg / L+6-BA0.11mg / L+NAA0.11mg / L+2,4-D0 .11mg / L+sucrose 30g / L+1.0% agar, pH is 5.9, dark culture, culture temperature is 25 ℃, obtain non-emb...

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Abstract

The invention provides a method for producing paclitaxel and a precursor baccatin III thereof by using cephalotaxus hainanensis non-embryogenic cell suspension culture. The method comprises the following steps: (1) culturing a cephalotaxus hainanensis explant on a callus induction culture medium to obtain a non-embryogenic callus; (2) carrying out proliferation culture on the non-embryogenic callus on a callus proliferation culture medium to obtain a cell cluster which grows rapidly and loosely; (3) inoculating the cell cluster to a cell suspension starting culture medium to be cultured to obtain a non-embryogenic suspension cell system of liquid culture; (4) standing and settling the non-embryogenic suspension cell system and inoculating sediment into a liquid proliferation culture medium to obtain suspended cells; and (5) carrying out centrifugal sedimentation on the suspended cells and removing supernatant; carrying out follow-up treatment on the sediment; and separating and purifying by using a liquid chromatography to obtain the paclitaxel and the precursor baccatin III thereof. The method has the characteristics of high production efficiency, no occupation of forest lands, no damages to natural resources, stable yield, high content of effective components and simple extraction process.

Description

technical field [0001] The invention relates to a method for producing paclitaxel and its precursor baccatin III by suspension culture of non-embryogenic cells of Torreya hainanensis. Background technique [0002] Cephalotaxus hainanensis. is an evergreen tree belonging to Cephalotaxaceae. Mainly produced in Hainan Island, it is also distributed in southwestern Guangdong, southeastern Guangxi, southeastern and western Yunnan, and southeastern Tibet. Scattered in valleys, streams or hillsides in mountainous rain forests or monsoon forests at an altitude of 700-1200 meters. Hainan rough torreya is the most southerly distributed species of the genus Trichopinia, and it is also a slow-growing tree species. Due to the narrow ecological distribution and human destruction, its distribution area is constantly shrinking. It was listed as the second-class national key protection and endangered in 1987. plant. [0003] The secondary metabolites of Torreya hainanensis are rich in a v...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/02
Inventor 乔飞江雪飞王荣香王琴飞丛汉卿钱丹赵小青尹俊梅
Owner TROPICAL CORP STRAIN RESOURCE INST CHINESE ACAD OF TROPICAL AGRI SCI
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