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Method for producing bulbus fritillariae cirrhosae total alkaloid by adopting cell mass suspension culture

A cell mass and suspension culture technology is applied in the field of using cell mass suspension culture to produce total alkaloids of Fritillaria fritillary, which can solve the problems of long cycle, low degree of differentiation, affecting secondary metabolites, etc., and can solve the problem of long production time. , The effect of sufficient nutrients to meet a large number of needs

Active Publication Date: 2010-02-03
薛刚
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In tissue culture, the conventional solid culture method has poor dispersion of cell aggregates in the medium, so the cell aggregates absorb nutrients less than suspension culture, which makes the growth of cell aggregates slow and the cycle is long
In addition, the general suspension culture is cultured with a single cell as an explant; although the division ability of a single cell is strong, the degree of differentiation is extremely low, which is not conducive to the production of secondary metabolites in the cultured cells, and a single cell cannot form a The mass transfer gradient also affects the production of secondary metabolites

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] (1) Acquisition of cell clumps: Cut the callus induced by the scale leaves into an appropriate size on the ultra-clean workbench and transfer to the medium MS+6-BA 2.0mg L that produces cell clumps -1 +NAA0.4mg·L -1 +30g·L -1 Sucrose+agar 6.5~7.0g·L -1 and cultured at 18-22°C, with light intensity of 1000-1800 lx, and 12 hours of light per day, cell clumps can be induced in 30 days;

[0015] (2) Suspension culture of cell clumps: Cut the induced cell clumps into 3-4mm size on the ultra-clean bench, insert MS+6-BA 2.0mg·L -1 +NAA0.2mg·L -1 + Ribavirin 5mg.L -1 +30g·L -1 In the liquid medium of sucrose, the inoculum size is 30g·L -1 , the culture temperature is 18-22°C, the light intensity is 600-1000lx, the light is 12 hours a day, and the shaking culture is carried out under the condition of the rotation speed of 120-140r / min. The value-added multiple can reach 5.08 times, and the alkaloid content of the regenerative cell mass is 0.139%;

[0016] (3) Subculture ...

Embodiment 2

[0019] Change the suspension medium in the step of Example 1(2) to MS+6-BA1.0mg L -1 +NAA0.2mg·L -1 + Ribavirin 20mg·L -1 + sucrose 30g·L -1 , the other steps were the same as in Example 1. After 30 days of culture, the multiplier of the cell aggregates was 2.96 times, and the wound on the surface of the cell aggregates was severely browned.

Embodiment 3

[0021] The inoculum size of the cell mass in the embodiment 1 (2) step is changed from 30g L -1 Change to 50g·L -1 , the other steps were the same as in Example 1. After 30 days of culture, the multiplication factor of the cell clusters was 2.26 times, indicating that too large an inoculum volume was not conducive to the proliferation of the cell clusters.

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Abstract

The invention discloses a method for producing bulbus fritillariae cirrhosae total alkaloid by adopting cell mass suspension culture. In the method, a bulbus fritillariae cirrhosae cell mass obtainedfrom induction culture is cut into a size of 3-4 mm and then grafted into a liquid culture medium for suspension culture, and finally a culture product which has high alkaloid content (which is 2.04 times that of market bulbus fritillariae cirrhosae bulb) and short growing period (which lasts only 24-28 days) is obtained. The method better solves the problems of long time, low crude drug yield, low effective component content and the like which exist in conventional bulbus fritillariae cirrhosae producing methods. In addition, the method can realize the aim of large scale and fast production of effective components of bulbus fritillariae cirrhosae crude drug.

Description

technical field [0001] The invention relates to a method for tissue culture of total alkaloids of Fritillaria sichuanensis, in particular to a method for producing total alkaloids of Fritillaria sichuanensis by suspension culture of cell masses. Background technique [0002] Fritillaria cirrhosa D.Don is a perennial herb of Liliaceae, whose bulbs are used as medicine. It is a commonly used drug for treating dry cough due to lung heat, dry cough with less phlegm, overworked cough due to yin deficiency, and bloody sputum. The main active ingredients are Fritillaria alkaloids and steroidal saponins. In nature, it takes about 4-5 years from the germination of Fritillaria chinensis seeds to the growth of medicinal materials, and the content of medicinal ingredients in them is very low, which has caused the over-excavation of wild Fritillaria sichuan resources, making the wild Fritillaria sichuan resources plummeting and is facing depletion. [0003] Fu Hualong and others obtai...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P1/00
Inventor 王跃华代勇何宗晟徐世军孙雁霞王晓蓉闫胜杰
Owner 薛刚
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