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Cylindrocarpon didymium and method for preparing ginsenoside Rh2 by using same

A technology of ginsenoside and twin columns, which is applied in the field of preparing rare ginsenoside Rh2, can solve the problems of poor selectivity, difficult separation of mixtures, economic impracticality, etc., and achieve the effect of low cost and few by-products

Inactive Publication Date: 2011-10-26
DALIAN NATIONALITIES UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Scholars in the former Soviet Union synthesized the compound by condensation of panaxatriol and acetylbromoglucose (new progress in the study of ginseng chemical constituents, "Chinese Journal of Medicinal Chemistry", 1992, 2 (1): 64-70.), but the selectivity is poor , the mixture is difficult to separate
Later, scholars from the former Soviet Union synthesized ginsenoside Rh through five-step regioselective and stereoselective reactions using betulintriol obtained from birch leaves as a raw material. 2 , but the reaction steps are cumbersome and economically impractical

Method used

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  • Cylindrocarpon didymium and method for preparing ginsenoside Rh2 by using same
  • Cylindrocarpon didymium and method for preparing ginsenoside Rh2 by using same
  • Cylindrocarpon didymium and method for preparing ginsenoside Rh2 by using same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: Isolation and screening of bacterial strains

[0038] 1. Isolation of ginseng pathogenic bacteria: Rinse the diseased fresh ginseng with tap water, blot the water with filter paper, then soak it with 0.1% mercury liter for 1-1.5 minutes, rinse it with sterile water for 4-5 times, and then use Soak in 75% alcohol for 1 to 1.5 minutes, and then rinse with sterile water for 3 to 4 times. After the treatment, under aseptic conditions, the ginseng root tissue was cut into small pieces (4mm×4mm) with sterilized surgical scissors, and placed on PDA plates with a diameter of 9cm. After 5-7 days, observe whether there are colonies around the tissue block. After the colonies are produced, pick mycelia and inoculate them on the PDA slant medium for purification and preservation for future use.

[0039] Medium: potato dextrose agar medium (PDA medium): 200 g of potatoes, 18 g of glucose, 18 g of agar, 1000 mL.

[0040] The ginseng (Panax ginseng C.A.Mey) used in th...

Embodiment 2

[0050] Embodiment 2: conversion control experiment

[0051] Adopt the method of embodiment 1 step 2 (2), the conversion selectivity of ginsenoside substrate and product of Cylindrospora bispora (CGMCCNo.4681) obtained by test screening, through TLC detection, the result is as attached image 3 Shown:

[0052] It can be seen that the protodiol saponin Rb 1 , Rb 2 . 1 , Rb 2 , Rc, and Rd all react, and for Rb 1 The conversion effect of is strong, and according to the Rf value, it can be preliminarily judged that the main product is Rh 2 .

Embodiment 3

[0053] Embodiment 3: solid transformation method transforms ginsenoside Rb 1 Preparation of Rh 2

[0054] ① Preparation of solid transformation medium (PDA): Each 100mL medium contains 20g of potato, 1.8g of glucose, 1.8g of agar, and ginsenoside Rb 1 ; Deionized water preparation, 121 ℃, 1.0kPa autoclave for 30min.

[0055] ② Spot the activated Cylindrospora bispora (CGMCC No.4681) to the above-mentioned 1 cultured on PDA medium at 25°C for 5-7 days.

[0056] ③Product extraction and separation: Soak the culture in n-butanol and fully extract; the extract is centrifuged at 10,000r / min for 1min, filtered through a microporous membrane with a diameter of 0.22μm, and the target product is separated by HPLC.

[0057] HPLC conditions: flow rate: 0.6mL / min; detection wavelength: 203nm; column temperature 35°C; mobile phase: A is acetonitrile, B is high-purity water;

[0058] Gradient elution mobile phase ratio:

[0059] 0min, A is 20%, B is 80%;

[0060] 18min, A is 40%, B is...

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Abstract

The invention relates to a pathogenic fungus of ginseng, in particular to Cylindrocarpon didymium (CGMCC NO.4681). The Cylindrocarpon didymium is capable of preparing ginsenoside Rh2 by converting substrate ginsenoside Rb1Rd. The method for preparing ginsenoside Rh2 by using the Cylindrocarpon didymium comprises the following steps: by adopting an in-situ conversion method, spot-inoculating the Cylindrocarpon didymium onto a PDA (Potato Dextrose Agar) culture medium containing ginsenoside Rb1 and / or Rd, and performing stationary culture at 25 DEG C for 5-7 days; or by adopting a microbial fermentation conversion method, inoculating the Cylindrocarpon didymium onto a liquid fermentation culture medium, and culturing at 28 DEG C for 5-7 days; and collecting the enzyme liquid, mixing with the ginsenoside Rb1 and / or Rd, and reacting at 40 DEG C for 24 hours. The technical scheme for producing ginsenoside Rh2 has the characteristics of high specificity, high simplicity and convenience, high safety and reliability, low cost and few byproducts. The purity of the fermented product Rh2 is above 85%, and the conversion rate can be up to 45% or above.

Description

technical field [0001] The invention relates to a ginseng pathogenic fungus, Cylindrocarpon didymium (CGMCC No.4681), and the transformation of ginsenoside Rb by using the fungus 1 Preparation of rare ginsenoside Rh with ginsenoside Rd 2 Methods. Background technique [0002] In recent years, tumor diseases have seriously endangered human health. At present, treatment mainly relies on surgery, radiotherapy, chemotherapy and biological methods. The purpose is to kill tumor cells through chemical drugs, promote their death or prevent proliferation. However, the cytotoxicity of most drugs lacks specificity. , often indiscriminately kill normal cells, especially the actively proliferating hematopoietic stem cells, damage to normal cells often leads to serious complications, affects the therapeutic effect and causes patient compliance. Therefore, finding high-efficiency and low-toxic tumor drugs from natural medicines has become a research hotspot in recent years. [0003] Ma...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12P33/20C12R1/645
Inventor 吕国忠张薇孙晓东
Owner DALIAN NATIONALITIES UNIVERSITY
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