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Method for increasing alkannin content of onosma paniculatum cultivation cell

A technology for culturing cells and comfrey, applied in the field of increasing the content of shikonin in the cultured cells of comfrey by ultrasonic waves, can solve the problems of few plant cells, unclear influence mechanism, etc., achieves easy operation, and improves effective secondary metabolites. The effect of content and expansion is strong

Inactive Publication Date: 2009-04-08
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many studies have shown that ultrasound can improve the biotransformation efficiency of enzymes and microbial cells in the reactor, but the mechanism of ultrasound in the biological reaction process is still not very clear. In most cases, it is believed that the mechanical stimulation of ultrasound strengthens the biotransformation process. Medium mass transfer and mixing, even improve the enzyme activity, make the intracellular metabolic reaction more vigorous
In the past, most of the research on the biological effects of ultrasound focused on human and animal cells and microorganisms, and there were few studies on plant cells.

Method used

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  • Method for increasing alkannin content of onosma paniculatum cultivation cell

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] 1) Subculture of Comfrey cells: The subculture medium is: MS basal medium + NAA 1.0mg / L+2,4-D 0.2mg / L, the culture conditions are solid culture, pH value 5.6, temperature 25 ±1℃, after inoculating the Lithospermum erythrorhizon cells in the subculture medium, culture in the dark;

[0021] 2) Inoculate the comfrey cells subcultured for 10 days in a 100 mL Erlenmeyer flask containing 40 mL of M10 liquid medium containing 1 mg / L kinetin (KT). The culture temperature is 25±1℃, and the rotation speed of the shaker 110r / min, dark culture;

[0022] 3) On the second day of culture, apply ultrasonic stimulation with a power of 80 W and a duration of 5 minutes to the suspension cultured cells. As shown in Figure 1, the cells are stimulated by ultrasonic waves and continue to be cultured until the 12-day culture cycle is completed.

[0023] 4) After the culture, the content of shikonin in the cells was determined to be 1.1%, which was 20% higher than the control group without any ultr...

Embodiment 2

[0025] 1) Subculture of Comfrey cells: The subculture medium is: MS basic medium + NAA 1.0 mg / L + 2,4-D 0.2 mg / L, the culture conditions are solid culture, pH 5.6, temperature 25 ±1℃, after inoculating the Lithospermum erythrorhizon cells in the subculture medium, culture in the dark;

[0026] 2) The 10 days of subcultured Comfrey cells were inoculated in a 100 mL Erlenmeyer flask containing 40 mL of M10 liquid medium containing 1 mg / L kinetin (KT). The culture temperature is 25±1℃, the rotating speed of the shaker is 110r / min, and the culture is dark;

[0027] 3) On the 4th day of culture, apply ultrasonic stimulation with a power of 100W and a duration of 2min to the suspension cultured cells. After the cells are stimulated by the ultrasonic waves, continue to culture until the 12-day culture cycle is completed;

[0028] 4) After the end of culture, the content of shikonin in the cells was determined to be 1.2%, which was 31% higher than that of the control group without any ult...

Embodiment 3

[0030] 1) Subculture of Comfrey cells: The subculture medium is: MS basic medium + NAA 1.0 mg / L + 2,4-D 0.2 mg / L, the culture conditions are solid culture, pH 5.6, temperature 25 ±1℃, after inoculating the Lithospermum erythrorhizon cells in the subculture medium, culture in the dark;

[0031] 2) The 10 days of subcultured Comfrey cells were inoculated in a 100 mL Erlenmeyer flask containing 40 mL of M10 liquid medium containing 1 mg / L kinetin (KT). The culture temperature is 25±1℃, the rotating speed of the shaker is 110r / min, and the culture is dark;

[0032] 3) On the 6th day of culture, apply ultrasonic stimulation with a power of 200W and a duration of 1 min to the suspension cultured cells. After the cells are stimulated by the ultrasonic waves, continue to culture until the 12-day culture cycle is completed;

[0033] 4) After the culture, the content of shikonin in the cells was determined to be 1.3%, which was 42% higher than that of the control group without any ultrasoun...

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Abstract

The invention relates to a method for increasing the alkannin content of the cultured cells of onosma paniculatum. The operating steps of the method mainly comprise: 1. the subculture of onosma paniculatum cells; 2. the process of liquid culture and ultra sonication of the onosma paniculatum cells; and 3. the process of measuring alkannin content. Utilizing ultrasonic to stimulate the suspension cultured onosma paniculatum cells is an effective means to increase the alkannin content of cells. The method is characterized by simple and easy operation, good effect and strong expansibility and can be applied to a large-scale bioreactor for plant cells to improve the content of the effective secondary metabolites.

Description

Technical field [0001] The invention relates to a method for increasing the content of shikonin in cultured cells of Lithospermum erythrorhizon using ultrasonic waves. Background technique [0002] Yunnan comfrey (Onosma paniculatum Bur.et Fr.) is mainly produced in Yunnan and is a commonly used Chinese herbal medicine in Yunnan Province. It belongs to the Boraginaceae (Boraginaceae) plant and its roots are used for medicinal purposes. The main active ingredient is shikonin and its derivatives. These ingredients not only have antibacterial, anti-inflammatory, anti-cancer and other pharmacological effects, but are also widely used as natural pigments in medicine, cosmetics and printing and dyeing industries. At present, the wild resources of comfrey are seriously damaged, and the contradiction between supply and demand in the market is prominent. The use of large-scale plant cell culture technology to directly cultivate cells in a reactor to obtain medicinal secondary metabolites ...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12P7/26
Inventor 葛锋刘畅刘迪秋陈朝银
Owner KUNMING UNIV OF SCI & TECH
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