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Method used for producing camptothecin and 10-hydroxycamptothecine with camptotheca acuminate suspension cells

A technology of hydroxycamptothecin and suspension cells, which is applied in the field of medicine and biology, can solve the problems of low product content, and achieve rapid growth and reproduction, simple and easy-to-control methods, and easy-to-cultivate effects

Inactive Publication Date: 2017-08-15
CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the problem of low product content in the existing production of camptothecin and 10-hydroxycamptothecin by camptothecin suspension cells, the present invention provides a method to increase the concentration of camptothecin and 10-hydroxycamptothecin by adding inducing factors during the cultivation of camptotheca suspension cells. Method for 10-Hydroxycamptothecin Yield

Method used

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  • Method used for producing camptothecin and 10-hydroxycamptothecine with camptotheca acuminate suspension cells
  • Method used for producing camptothecin and 10-hydroxycamptothecine with camptotheca acuminate suspension cells
  • Method used for producing camptothecin and 10-hydroxycamptothecine with camptotheca acuminate suspension cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Establishment of camptophylla aseptic seedlings

[0025] 1. Test material

[0026] 1.1 Plant material

[0027] Choose the amphocarpa fruit that grows normally and healthy, and peel, clean, and disinfect according to routine operations. In the present embodiment, the seed coat of the fruit is first removed, then soaked with 5% Triton for 3 minutes, rinsed repeatedly with sterile water, the concentration is 75% ethanol for 1 minute, 1% sodium hypochlorite solution for 3 minutes, and sterile water for 10 times. 1min, and finally use sterile filter paper to absorb the moisture on the surface of Campylocarpa seeds, and set aside.

[0028] 1.2 Medium

[0029] 1 / 2 MS solid medium: add 0.35% agar (Sigma Chemical, MO) to 1 / 2 MS medium, pH 5.8, autoclave at 121° C. for 30 min.

[0030] 2. Inoculation of seeds into culture medium

[0031] The prepared seeds were inoculated and cultivated according to conventional aseptic methods.

[0032] Inoculate the prepared se...

Embodiment 2

[0033] Embodiment two: the establishment of camptophylla callus

[0034] 1 test material

[0035] 1.1 Plant material

[0036] The aseptic seedling that obtains among the embodiment one. According to routine aseptic operation, cut the leaves into small pieces of 10 mm×10 mm with a sterile scalpel, and set aside.

[0037] 1.2 Medium

[0038] 7 kinds of solid medium based on MS30 (30mg / L sucrose added to MS medium) and different compositions and concentrations of growth regulators. As in Table 1:

[0039] Table 1 Composition of Medium A-J

[0040]

[0041] 2. Inoculation of sterile leaves into culture medium

[0042] The prepared leaves were inoculated and cultured according to conventional aseptic methods.

[0043] Inoculate the prepared leaves on the corresponding medium in Table 1, and culture them in the dark at 25°C for 5-6 weeks until the callus grows. Three parallel experiments were performed on different media.

[0044] 3. Preparation of Metabolites from Campylo...

Embodiment 3

[0057] Example 3: Establishment of Campylocarpa suspension cell line

[0058] 1 test material

[0059] 1.1 Plant material

[0060] The Campylocarpa callus obtained in Example 2 was cut into small pieces with a sterile scalpel according to conventional aseptic procedures, and was set aside.

[0061] 1.2 Medium

[0062] Add 0.5mg / L NAA and 2mg / L BAP to MS30 liquid medium.

[0063] 2. Inoculation of Campylodon japonica callus into culture medium

[0064] The prepared callus was inoculated and cultured according to conventional aseptic methods.

[0065] The callus was inoculated into 100mL liquid medium and cultured under continuous light. Light culture conditions: white light (25 μmol m -2 the s -l ), the temperature is 25° C., the humidity is 70%, and the rotation speed is 125 rpm. After 3 to 5 days, when cells grow out of the culture medium, absorb 5 mL of the cell suspension and transfer it to 100 mL of a new liquid medium, and culture under the same conditions to obta...

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Abstract

The invention relates to a method used for producing camptothecin and 10-hydroxycamptothecine with camptotheca acuminate suspension cells, and belongs to the field of medical biotechnology. The method comprises following steps: 1, MS liquid medium is inoculated with camptotheca acuminate suspension cells for culturing, wherein sorbitol is added in culturing process; 2, corresponding suspension cells and cell culture fluid are collected; and 3, separation extraction of camptothecin and 10-hydroxycamptothecine are carried out. The method used for producing camptothecin and 10-hydroxycamptothecine with camptotheca acuminate suspension cells is simple, and is convenient to control. Compared with a conventional camptothecin and 10-hydroxycamptothecine preparation method, the method comprises following advantages: sorbitol is taken as an inducible factor, and is capable of increasing camptothecin and 10-hydroxycamptothecine yield by more than 100 to 500 times.

Description

technical field [0001] The invention relates to a method for producing camptothecin and 10-hydroxycamptothecin by using camptothecin suspension cells, belonging to the field of medical biotechnology. Background technique [0002] Camptotheca acuminate, also known as eclipta, water chestnut, water tung tree, celestial catalpa, eclipta, thousand-foot tree, wild plantain, water mozi, is a plant of the genus Camptotheca in the family Davidiaceae. It is mainly distributed in the south of the Yangtze River in China. In August 1999, with the approval of the State Council of the People's Republic of China, camphor tree was listed as the first batch of national key protected wild plants, and the protection level was Ⅱ. [0003] Campylodida has good medicinal value, and its fruit, bark, roots, branches, and leaves can be used as medicine. Modern studies have shown that the main medicinal active ingredients in camphor trees are alkaloids. Clinically, camptothecin is mostly extracted...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/18C12N5/04
CPCC12N5/04C12N2500/35C12P17/18
Inventor 罗应刚蒲祥杨云屈细兴陈菲张国林
Owner CHENGDU INST OF BIOLOGY CHINESE ACAD OF S
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