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Biotransformation and purification method of 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin

A technique for demethylepodophyllin and tetramethylpyrazine, which is applied in the field of biotransformation of podophyllotoxins, can solve the problems of high price, high production cost, small scale and the like, and achieves simple operation, low cost and short cycle. Effect

Inactive Publication Date: 2013-10-16
HUBEI UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, with the increasing demand of human beings, the chemical synthesis method has shown great disadvantages: first, the scale is small and the output is low; second, the chemical residues are difficult to remove; third, the production cost is high
These above-mentioned factors will inevitably cause the price of 4-(2,3,5,6-tetramethylpyrazin-1-yl)-4'-norepipodophyllotoxin to be high, so it is necessary to find a method suitable for large-scale industrial production. The method of 4-(2,3,5,6-tetramethylpyrazin-1-yl)-4'-norepipodophyllotoxin into 4-(2,3,5,6-tetramethylpyrazine Zin-1-yl)-4′-norepipodophyllotoxin research and development is the most important

Method used

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  • Biotransformation and purification method of 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin
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  • Biotransformation and purification method of 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] 1. In the first stage, podophyllotoxin is converted into 4′-desmethyl epipodophyllotoxin:

[0073] 1), slant strain cultivation: the formula of the slant strain culture medium adopted in the present embodiment is (nutritional PDA slant): glucose 30 grams / liter, magnesium sulfate 0.5 grams / liter, potassium dihydrogen phosphate 1.5 grams / liter, Thiamine hydrochloride 0.1 g / L, agar 20 g / L, potato extract 1.0 L; the preparation method of potato dipping juice: 200g fresh potatoes are peeled and cut into square pieces with a side length of about 1cm, and the cut pieces Put it in an enamel tank with 1L of deionized water, boil for 15 minutes, and when the potatoes are soft and not loose, filter them with eight layers of gauze, take the filtrate and dilute it to 1L again, and the obtained solution is used to prepare PDA medium.

[0074] Gibberella Fujikura SH-f13 was inoculated on the PDA slant by the two-point method with an inoculation needle, and the culture conditions were ...

Embodiment 24

[0097] Example 2 Separation and purification of 4-(2,3,5,6-tetramethylpyrazin-1-yl)-4'-norepipodophyllotoxin

[0098] (1), prepare the sample to be separated: take the Alternaria tenospora mycelium obtained in Example 1 and place it in a 60°C oven for 4 days, and after the mycelium is completely dry, grind it into a powder sample with a pulverizer, and take 200 grams This sample is in the round bottom flask of 2 liters and adds a small amount of broken ceramic pieces, adds 1 liter of chloroform, the upper end of the round bottom flask is connected with the reflux extractor, and the upper end of the extractor is connected with the condensing pipe cold water reflux ( figure 2 ). Heating to boiling and maintaining for 2 hours ended as one time, and reflux extraction was performed three times. The filtered chloroform was rotary evaporated, and the sample was vacuum-dried, and 5 ml of chloroform was taken to reconstitute the dried sample, and stored in a refrigerator at 4°C as my...

Embodiment 34

[0102] Example 34-(2,3,5,6-tetramethylpyrazin-1-yl)-4'-norepipodophyllotoxin content determination

[0103] (1), the fermentation broth obtained in Example 1 was centrifuged (10000 rpm, 20 minutes, 4°C), and 2 ml of supernatant was used for 4-(2,3,5,6-tetramethylpyridine Determination of oxazin-1-yl)-4'-norepipodophyllotoxin; 4 ml of acetonitrile was added to 2 ml of fermentation broth, and centrifuged (13000 rpm, 20 minutes); the supernatant was passed through a 0.45 micron needle Filter through a filter membrane and store in a refrigerator at 4°C as a test sample of the fermentation broth.

[0104] (2), take the fresh mycelium obtained in Example 1 and dry it at 60°C, grind the dried mycelium to 200 mesh powder, take 100 grams of mycelium powder in a 2-liter round bottom flask and add A small amount of broken ceramic pieces, add 1 liter of chloroform, the upper end of the round bottom flask is connected to the reflux extractor, and the upper end of the extractor is connecte...

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Abstract

The invention discloses a method for transforming podophyllotoxin into 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin, comprising the following steps of: (1) adding podophyllotoxin into a liquid fermentation medium, and carrying out biotransformation by inoculating second-class liquid seeds of humic soil fungi; (2) separating and purifying the product from the Step (1), followed by adding into the liquid fermentation medium, carrying out biotransformation by inoculating second-class liquid seeds of podophyllum taxa endophytes; then continuously carrying out biotransformation by adding ligustrazine, and collecting the transformation product. The invention also discloses a method for purifying 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin from the above product and a method for detecting the content of 4-(2,3,5,6-tetramethylpyrazine-1-group)-4'-demethylepipodophyllotoxin. According to the invention, the molecular structure of podophyllotoxin is modified by the biotransformation method, which has advantages of mild reaction condition, low E-factor, quality controllability and simple separation process.

Description

technical field [0001] The present invention relates to a preparation method of podophyllotoxin, in particular to a biotransformation method of 4-(2,3,5,6-tetramethylpyrazin-1-yl)-4'-norepipodophyllotoxin , the present invention also relates to the separation and purification method and content detection method of the 4-(2,3,5,6-tetramethylpyrazin-1-yl)-4'-norepipodophyllotoxin, belonging to the podophyllotoxin class The field of biotransformation of compounds. Background technique [0002] 4-(2,3,5,6-tetramethylpyrazin-1-yl)-4′-norepipodophyllotoxin is a podophyllotoxin (also known as 4-(2,3,5, 6-tetramethylpyrazine-1)-4'-demethylepipodophyllotoxine), its structural formula is shown in figure (I). [0003] [0004] (I) [0005] For the 4th position of the C ring of podophyllotoxin, the derivatives obtained by substituting the β carbon-nitrogen bond aromatic ring as the modification direction generally have good antitumor activity and bioavailability,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P17/16C07D493/04G01N30/02G01N30/08C12R1/645
Inventor 汤亚杰赵巍
Owner HUBEI UNIV OF TECH
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