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Method for improving tanshinone content in salvia miltiorrhiza hair roots through methyl jasmonic acid treatment

A technology of tanshinone content and methyl jasmonic acid, applied in horticultural methods, botany equipment and methods, horticulture, etc., can solve problems such as quality degradation, long growth cycle of salvia miltiorrhiza, environmental pollution, etc., to increase the content of tanshinone and facilitate scale The effect of streamlining production and reducing production costs

Inactive Publication Date: 2012-07-18
开国银
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main source of tanshinone is extracted from the underground part of the root of Danshen. However, the long growth cycle of Danshen and the serious degradation of its quality have become a drawback of the traditional cultivation of Danshen
The complex structure of tanshinone makes its chemical synthesis process very cumbersome, high cost and easy to cause environmental pollution; cells obtained by cell culture methods under in vitro conditions have low accumulation of active components and poor stability
In addition, it is a more feasible method to use genetic engineering technology to obtain new varieties of salvia miltiorrhiza with higher tanshinone content, but it is still in the experimental stage to use genetic engineering to increase tanshinone content.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Salvia miltiorrhiza infecting with Agrobacterium rhizogenes to obtain hairy roots of Salvia miltiorrhiza

[0014] 1.1 Pre-cultivation of explants

[0015] Cut the leaves (0.5 cm2) of robust aseptic seedlings of Salvia miltiorrhiza, inoculate them on the pre-cultivation medium (MS), and culture them in the dark at 25°C for 2 days.

[0016] 1.2 Co-cultivation of Agrobacterium and explants

[0017] Put the above-mentioned explants of pre-cultivated leaves of Salvia miltiorrhiza into the 1 / 2MS suspension containing the activated Agrobacterium rhizogenes engineering bacteria and soak for 10 minutes (shaking gently to make the explants fully contact with the bacterial solution) , take out the dipped Salvia miltiorrhiza leaves, blot the surface bacterial liquid with sterile absorbent paper, transfer to co-cultivation medium 1 / 2MS, and culture in dark for 3-4 days.

[0018] 1.3. Induction and subculture of hairy roots

[0019] Transfer the above-mentioned salvia explants co-...

Embodiment 2

[0024] Induction treatment of hairy roots of Salvia miltiorrhiza with methyl jasmonic acid

[0025] 2.1. Liquid culture of hairy roots

[0026] Select the hairy roots that grow fast and branch well in Example 1, cut 2-3cm on the ultra-clean workbench and rinse off the agar on its surface with sterile water distilled water and then insert the 100ml 1 / 2MS liquid culture 100rpm in a culture bottle with medium, cultured in the dark at 25°C, and replaced with fresh 1 / 2MS liquid medium for subculture every 20 days. Get a certain amount of hairy roots of Salvia miltiorrhiza that have grown well in liquid medium through 3 times of secondary culture (about 2 months old) and re-inoculate in multiple 250ml shake flasks with 100ml 1 / 2MS liquid medium cultured as experimental material. Suspension culture conditions are 25°C, 100r / min, dark culture.

[0027] 2.2 Induction treatment of hairy root of Salvia miltiorrhiza

[0028] From the cultivation of the hairy roots of Salvia miltiorrhi...

Embodiment 3

[0030] Determination of Tanshinone Content in Salvia Miltiorrhiza Hairy Roots by HPLC

[0031] 3.1. Chromatographic conditions and preparation of standard stock solutions

[0032] The chromatographic column is a C-18 reverse-phase silica gel column, the mobile phase is acetonitrile:water (65:35), the detection wavelength is 270nm, the column temperature is 30°C, the flow rate is 1ml / min, and the injection volume is 20μl.

[0033] Precisely weigh cryptotanshinone, tanshinone I, and tanshinone II A standard products to prepare stock solutions with concentrations of 36 μg / ml, 36 μg / ml, and 22 μg / ml respectively, and store them at -20°C for later use.

[0034] 3.2. Preparation of standard curve

[0035] In the present invention, when the mobile phase acetonitrile: water is at a volume ratio of 65:35, the retention times of cryptotanshinone, tanshinone I, and tanshinone II A are respectively 18.90 min, 20.53 min and 34.00 min, and the three tanshinone components are completely sep...

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Abstract

The invention belongs to the technical field of biological culture, in particular to a method for improving the tanshinone content in salvia miltiorrhiza hair roots through methyl jasmonic acid treatment. According to the method, agrobacterium rhizogene is firstly used for infecting salvia miltiorrhiza aseptic seedlings to obtain the salvia miltiorrhiza hair roots, then, the sterilization culture of the salvia miltiorrhiza hair roots is carried out, and next, the salvia miltiorrhiza hair roots are selected to be transferred into a liquid culture medium for carrying out suspension culture; and methyl jasmonic acid is added into the salvia miltiorrhiza hair roots in the time of 4 to 6 days before the salvia miltiorrhiza hair root liquid culture harvesting, then, the harvesting is carried out on time, and the tanshinone content in salvia miltiorrhiza hair roots can be greatly improved. The method provided by the invention has the advantages that the tanshinone content in the hair roots can be effectively improved, the operation is simple, the relatively higher front-stage investment required by the utilization of a gene engineering method for improving the tanshinone content is not needed, the production cost of the tanshinone is effectively reduced, and great significance is realized on meeting the large-scale production of the tanshinone.

Description

technical field [0001] The invention belongs to the technical field of biological cultivation, and in particular relates to a method for increasing the content of tanshinone in hairy roots of salvia miltiorrhiza. Background technique [0002] Salvia miltiorrhiza Bunge, also known as purple salvia, red root, red ginseng, blood ginseng, etc., belongs to the genus Salvia of the Labiatae family, and is a perennial herb. As a traditional Chinese medicine, Danshen is mainly used clinically in the treatment of cardiovascular diseases. The main medicinal components of Danshen include fat-soluble diterpene quinones (tanshinones) and water-soluble phenolic acids. Modern studies have shown that the tanshinone substances contained in Salvia miltiorrhiza have various pharmacological activities such as anti-tumor, anti-bacterial and anti-inflammatory, anti-oxidation, and anti-atherosclerosis, so there is a huge market demand. At present, the main source of tanshinone is extracted from t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
Inventor 开国银
Owner 开国银
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