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Technical method for synthesizing 10-hydroxy camptothecin by artificially inducing icacinaceae endophyte

A technology of hydroxycamptothecin and stinky horse than wood, applied in the biological field, can solve the problems of low conversion rate, high technical requirements for strain cultivation, and many separation steps, and achieve the effects of low cost, easy industrial production, and easy cultivation

Inactive Publication Date: 2011-06-01
张苑金
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This scheme has certain economic benefits, but there are still disadvantages such as low conversion rate, high technical requirements for strain cultivation, and many separation steps.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] (1) Medium preparation: Add 50 μmol / L of naphthaleneacetic acid, 5 μmol / L of 6-benzylaminopurine, 30 g / L of sucrose or glucose, and 1.5 g / L of agar into MS basic medium; pH5.5~6;

[0013] (2) Inoculation culture: place the immature cells of Sinomabi wood at a ratio of 100 g / L wet weight, place them on a shaker at 25°C, and cultivate them in the dark at a speed of 80 rpm;

[0014] (3) Combined elicitor: composed of 0.02mol endophyte elicitor, 60g / L salicylic acid, 20μl / L hydrogen peroxide and 80μmol / L methyljasmonic acid;

[0015] (4) Combined induction: During the cultivation process, the inducer was used to cultivate, and 5 days after the inoculation of the medium, an inducer with a concentration of 300 μmol / L was added, and 30 g / L of sugar was added; 10 days after the inoculation of the medium, a concentration of 500 μmol / L combined elicitor;

[0016] (5) Extraction: Separating from the obtained culture, extracting 10-hydroxycamptothecin from the cell part and the ad...

Embodiment 2

[0018] (1) Medium preparation: Add 50 μmol / L of naphthaleneacetic acid, 5 μmol / L of 6-benzylaminopurine, 30 g / L of sucrose or glucose, and 1.5 g / L of agar into MS basic medium; pH5.5~6;

[0019] (2) Inoculation and cultivation: place the immature cells of stinky horsebiwood in a 25°C shaker at a speed of 100rpm according to the ratio of the inoculation amount to 100g / L wet weight, and culture in the dark;

[0020] (3) Combined elicitor: composed of 0.02mol endophyte elicitor, 60g / L salicylic acid, 20μl / L hydrogen peroxide and 80μmol / L methyljasmonic acid;

[0021] (4) Combined induction: During the cultivation process, the inducer was used to cultivate, and 7 days after the inoculation of the medium, an inducer with a concentration of 600 μmol / L was added, and 30 g / L of sugar was added; 10 days after the inoculation of the medium, a concentration of 1000μmol / L combined inducer;

[0022] (5) Extraction: Separated from the obtained culture, 10-hydroxycamptothecin in the cell pa...

Embodiment 3

[0024] (1) Medium preparation: Add 60 μmol / L of naphthaleneacetic acid, 5 μmol / L of 6-benzylaminopurine, 30 g / L of sucrose or glucose, and 1.5 g / L of agar into MS basic medium; pH5.5~6;

[0025] (2) Inoculation and cultivation: place the immature cells of Sinomabi wood at a rate of 150g / L wet weight in a shaker at 25°C with a rotation speed of 100rpm, and culture in the dark;

[0026] (3) Combined elicitor: composed of 0.03mol endophyte elicitor, 70g / L salicylic acid, 20μl / L hydrogen peroxide and 80μmol / L methyljasmonic acid;

[0027] (4) Combined induction: During the cultivation process, the inducer was used for cultivation. Seven days after inoculation of the medium, an inducer with a concentration of 600 μmol / L was added, and 40 g / L of sugar was added; 15 days after the inoculation of the medium, a concentration of 1200μmol / L combined inducer;

[0028] (5) Extraction: Separated from the obtained culture, 10-hydroxycamptothecin in the cell part and the adsorbed part was ex...

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Abstract

The invention relates to a technical method for synthesizing 10-hydroxy camptothecin by artificially inducing icacinaceae endophyte. The method comprises the following steps of: filling immature cells of icacinaceae in an amplification culture medium according to a proportion of inoculum size to wet weight of between 50 and 200g / L and culturing at the temperature of between 25 and 30 DEG C; adding combined inducer at the concentration of between 1 and 2,000 mu mol / L and sugar at the concentration of between 20 and 50g / L at the cell index growth initial stage; and adding the combined inducer at the concentration of between 1 and 2,000 mu mol / L at the cell index growth medium stage for induction. In the method, the characteristics that the endophyte is easy to culture and control and fast in growth, and the like are utilized, the 10-hydroxy camptothecin which has the same physiological activity as a plant source can be produced in an artificial induction mode in combination with the characteristics of the icacinaceae which is used as a raw material for producing the camptothecin; the method has the advantages of low cost and easiness in industrial production; and a new approach can be developed for searching anti-cancer medicaments in view of microorganisms.

Description

technical field [0001] The invention relates to a technical method for synthesizing camptothecin, in particular to a technical method for synthesizing 10-hydroxycamptothecin by artificially inducing endophytic bacteria of P. stifolium, belonging to the field of biotechnology. Background technique [0002] 10-Hydroxy camptothecin (10-Hydroxy camptothecin, referred to as HPT) was originally a new type of effective anti-tumor drug extracted from the fruit of the unique plant Camptothecin in my country. Intermediates of important camptothecin derivatives such as Irinotecan and Topotecan. Because the anti-cancer effects of the new anti-cancer drugs "Inotecan" and "Topotecan" are generally equivalent to or better than those of paclitaxel (actually in the early 1990s, clinical trials organized by authoritative medical research institutions designated by the US FDA showed that ), therefore, camptothecin and its derivatives have attracted widespread attention as an important antitumo...

Claims

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Application Information

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IPC IPC(8): C12P17/18
Inventor 张苑金
Owner 张苑金
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