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Method for production of phytolaccatoxin medicinal active substance

A kind of technology of active ingredient, terus saponin

Inactive Publication Date: 2003-10-15
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method has some deficiencies in terms of induction efficiency, sterilization methods, etc., and the content of active ingredients in hairy roots has not been investigated and strains with high content of active ingredients have not been screened.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment one: the acquisition of pokeweed hairy root

[0042] 1.1 Activation of Agrobacterium rhizogenes, three Agrobacterium rhizogenes strains A4, R1600, and C58C1 were stored in sterilized glycerol at -20°C, activated 3 times in YEB solid medium at 25°C before use, and then transferred to YEB culture solution (200mg / L kanamycin was added to the culture solution) was cultured in the dark at 25°C with 100r / min shaking, and after about 12 hours, the absorbance (OD) at 600nm of the bacterial solution was measured. 600 value), when OD 600 A value of 0.8 was used to infect explants.

[0043] 1.2 Plant material, take pokeweed seeds, soak them in concentrated sulfuric acid for 2 hours, wash them with water, soak them in 75% ethanol for 1 min, rinse the ethanol with water, and wash them with 0.1% HgCl 2 Disinfect for 15 minutes, rinse with sterile water 5 times, place on MS medium, 25°C, light 12h / d, light intensity 2000lx, germinate after 15d. The cotyledons and hypocot...

Embodiment 2

[0051] Embodiment two: the detection of hairy root

[0052] 2.1 PCR method

[0053] DNA from hairy roots was extracted by the phenol-chloroform method. The rolB and rolC genes are key genes related to transformation in Agrobacterium rhizogenes, and the PCR primers for these two genes were designed and synthesized respectively.

[0054] The primers for the rolB gene are

[0055] 5'-GCTCTTGCAGTGCTAGATTT-3' and 5'-GAAGGTGCAAGCTACCTCTC-3',

[0056] The expected product length is 423bp,

[0057] The primers for the rolC gene are

[0058] 5'-CTCCTGACATCAAACTCGTC-3' and 5'-TGCTTCGAGTTATGGGTACA-3',

[0059] The expected product length is 626bp.

[0060] The PCR reaction system is 25μl (50ng genomic DNA, 50mM KCl, 10mM Tris-HCl (pH8.3), 1.5mM MgCl 2 , 200 μM dNTPs, 1.25 units Taq DNA polymerase, 25 pmol primers). The reaction conditions were denaturation at 94°C for 3 minutes, 35 cycles (denaturation at 94°C for 40 seconds, annealing at 55°C for 40 seconds, extension at 72°C fo...

Embodiment 3

[0063] Embodiment three: the mensuration of medicinal active ingredient in hairy root

[0064] 3.1 Determination of the content of pokeweed saponin (esculentoside) (sulfuric acid-vanillin colorimetric method)

[0065] Precisely weigh 5 mg of Phytolaside A standard substance dried to balance, put it in a 10ml volumetric flask, dissolve it with methanol and constant volume, measure 0.2, 0.4, 0.6, 0.8, 1.0ml respectively, put it in a stoppered test tube, add water to set To a volume of 1ml, add 2.0ml of 8% vanillin ethanol solution, 5.0ml of 77% sulfuric acid solution, mix well and place at 60°C for 10 minutes, then place on ice for 15 minutes, use 1.0ml of water as a blank, and measure at 450nm The optical density was used to obtain a standard curve.

[0066] Pound the dried herbs and dried hairy roots into powder, pass through a 40-mesh sieve, bake at 60°C for 2 hours, accurately weigh 2 grams, put methanol in a Soxhlet extractor to reflux until there is no saponin, and recove...

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Abstract

The production process of medicinal active component phytolaccatoxin belongs to medicine technology and biological technology. The process includes conversion with cotyledon and plumular axis sprouting for seven days as inducing material; infecting explant in bacteria fluid, bateria-free water washing and culture in dark place for two days; secondary culture for at least five times in culture medium with Cedfotaxime to eliminate agrobacterium or high temperature sterilizing through transferring hairy root into thermotank at 39.5 deg.c to culture for 48 hr; separating hairy roots one by one, and culture in 1 / 2MS culture liquid in large amounts to produce phytolaccatoxin and pokeberry polysaccharide. The present invention has the advantages of high yield and stable content.

Description

technical field [0001] The invention relates to a method for producing the medicinal active ingredient of pokeweed saponin, discloses a method for producing the medicinal active ingredient of pokeweed saponin by using the hairy root of pokeweed and its culture solution, and belongs to the fields of medical technology and biotechnology. Background technique [0002] Phytolacca acinosa Roxb (Phytolacca acinosa Roxb) is a Phytolacca family plant, and its root is an important traditional Chinese medicine, which is produced in most parts of my country. Phytolacca was first recorded in "Shen Nong's Materia Medica", and was listed as inferior because of its toxicity. Clinically, it is mostly used for edema, fullness, constipation, and external treatment of pain, swelling and sore. Modern pharmacological studies have shown that pokeweed saponins have the activity of activating nucleotide reductase, pokeweed total saponins can induce R interferon, glycoproteins can inhibit the DNA s...

Claims

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Application Information

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IPC IPC(8): A61K31/706A61P1/10A61P43/00C07H17/02C12N5/04
Inventor 许铁峰唐克轩张汉明张磊
Owner SHANGHAI JIAO TONG UNIV
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