Arnebia root endophyte as well as screening method, culture method and application thereof

A screening method, the technology of comfrey root, applied in the field of microorganisms, can solve the problems of unclear source of anti-colon cancer efficacy of comfrey root, unfavorable production of anti-colon cancer drugs, long planting time period and other problems, and achieve obvious anti-colon cancer effect, Potential medicinal value, good economic benefits

Pending Publication Date: 2022-05-27
南京合谷生命生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Chinese patent CN201910725295.1 discloses a method for extracting and separating the active ingredients of comfrey root and the application of its extract in the preparation of drugs for inhibiting the growth of colon cancer cells. It is reported that the extract of comfrey root has anti-colon cancer effect by means of plant extraction. However, the source of the anti-colon cancer effect of comfrey root is still unclear, and plant extraction consumes a large amount of comfrey root medicinal materials, and the planting period is long, which is greatly affected by the season, which is not conducive to the production of anti-colon cancer drugs.
In addition, some studies have shown that the endophytic bacteria of the root of the comfrey is an ecological group that has a mutually beneficial symbiotic relationship with the root of the comfrey, which can promote the growth and metabolism of the host plant, enhance the antibacterial ability, and is a natural part of the plant micro-ecosystem. of endophytes remain undiscovered

Method used

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  • Arnebia root endophyte as well as screening method, culture method and application thereof
  • Arnebia root endophyte as well as screening method, culture method and application thereof
  • Arnebia root endophyte as well as screening method, culture method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1 Screening method

[0035] This embodiment provides a screening method for endophytes of comfrey roots: wash the comfrey roots with running tap water, use filter paper to absorb or dry the water on the comfrey roots, soak them in 75% ethanol for 3 minutes, and then soak them in 75% ethanol for 3 minutes. Soak the bacteria water three times for 3 minutes each time, then use 5% sodium hypochlorite solution to soak for 3 minutes, and finally use sterile water to wash 3~4 times to thoroughly clean the disinfectant. Aseptic scissors cut the comfrey root into thin sections of about 1 cm, and then placed it in a sterilized mortar for grinding and pulverizing, adding 1 ml of sterile water and mixing, and then moved to a shaking tube containing 5 ml of LB liquid medium. Then put it in a shaker for shaking culture, set the shaker temperature to 37°C and the rotation speed to 180rpm, incubate for 2~6h, take it out, let it stand for 5~10min, take 100μl of the upper liquid,...

Embodiment 2

[0036]Example 2 Identification of single colony species

[0037] Get the bacterial classification that has been preserved in the -80 ℃ refrigerator in the embodiment 1, activate the bacterial classification in the 100ml LB liquid medium according to the inoculation amount of 1% by volume, place the shaking culture in the shaking table, and set the temperature of the shaking table to be 37 ℃. ℃, the rotation speed is 225rpm, culture for 16h, after culture, put the bacterial liquid in a centrifuge tube, centrifuge in a refrigerated centrifuge, collect the bacterial species, use a genome extraction kit to extract the total genome DNA of the strain, and use the 16SrDNA universal primer for bacteria, Using the extracted genome as a template, PCR amplification was performed by denaturing at 95°C for 1 min, annealing at 50-60°C for 30s, and extending at 72°C for 2-5 min. The cycle was repeated 35 times. After PCR amplification, 1% agarose gel electrophoresis was performed. Electropho...

Embodiment 3

[0042] Example 3 The method of expanding the culture of strains

[0043] Get the comfrey root endophyte bacterial classification that has been preserved in -80 ℃ of refrigerators in embodiment 1, activate the bacterial classification in 100ml LB liquid medium according to the inoculation amount of 1% by volume, place in the shaker and carry out expanded culture, Set the shaker temperature to 37°C, the rotation speed to 225rpm, and cultivate for 12~24h, then place the fermentation broth in a centrifuge tube and use a refrigerated centrifuge for centrifugation. 5~12min, then remove the supernatant, collect the strain, then add 30ml of sterile PBS liquid, and then place it in a refrigerated centrifuge for centrifugation, remove the supernatant, and wash and remove the residual fermentation broth to obtain comfrey root endophytes. bacterial strains.

[0044] In order to identify the anti-colon cancer activity of comfrey root endophyte, the present invention prepares the comfrey r...

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Abstract

According to the alkanet endophyte, alkanet is used as a raw material, a strain is obtained through soaking and grinding, LB liquid culture medium shake culture, LB plate culture medium inverted culture, LB solid culture medium streak culture and LB liquid culture medium overnight culture screening in sequence, and the biological species of the alkanet endophyte are determined through identification; the strain is subjected to amplification culture through activation inoculation to obtain a bacterial strain, and identification shows that the alkanet endophyte has anti-colon cancer activity. The alkanet root endophyte is simple in obtaining mode, time is shortened, and the alkanet root endophyte can be applied to preparation of anti-colon cancer drugs.

Description

technical field [0001] The invention relates to a comfrey root endophyte and a screening method, a culture method and an application thereof, and belongs to the technical field of microorganisms. Background technique [0002] Comfrey Root is the dry root of the perennial herb of the comfrey family, Xinjiang comfrey Xinzang, false comfrey or Yunnan comfrey. The types of comfrey roots are mainly divided into soft comfrey roots and hard comfrey roots. The soft comfrey roots are irregular long cylindrical and twisted, mainly including Inner Mongolia comfrey; the hard comfrey roots are conical or cylindrical, twisted, including old strips. Lithospermum, Guan Lithospermum, Changde Lithospermum, etc.; Lithospermum root has the effects of clearing heat and cooling blood, deciphering rash, relaxing bowels, anti-tumor, anti-inflammatory, anti-pathogenic microorganisms, analgesic, and sedative; according to reports, scientists The anti-colon cancer effect of comfrey root was prelimina...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/02C12Q1/686C12Q1/689A61K35/74A61P35/00C12R1/01
CPCC12N1/20C12N1/02C12Q1/686C12Q1/689A61K35/74A61P35/00C12Q2565/125
Inventor 丁叶华夏李亚
Owner 南京合谷生命生物科技有限公司
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