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Technical process for synthesizing camptothecin sugar derivative by artificially inducing nothapodytes nimmoniana (graham) mablerley endophyte

A technology of camptothecin sugar and camptothecin sugar, which is applied in the field of artificially inducing camptothecin sugar derivatives and camptothecin derivatives by artificially inducing endophytic bacteria

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

[0004] Stinky mabi wood is another major source of camptothecin besides camptothecin in recent years. It has attracted attention because of its rich content of camptothecin. Related research has been carried out, such as the patent "separation from mabi wood Camptothecin skeleton compound and its use as a synthon of new drugs and therapeutic agents” (ZL97194492.X), “Methods for extracting camptothecin from S. Development and Utilization of Alkali", Feng Jiancan et al., "Forestry Science", 2000, Volume 36, No. 5), etc., but there are few reports on the synthesis of camptothecin and its derivatives by artificially inducing endophytic bacteria of S.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] (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;

[0014] (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;

[0015] (3) Combined elicitor: composed of 0.02mol endophyte elicitor, 60g / L salicylic acid, 20μl / L hydrogen peroxide and 80μmol / L methyljasmonic acid;

[0016] (4) Combined induction and saccharification reaction: During the cultivation process, the inducer was used to cultivate, and 5 days after inoculation of the medium, an inducer with a concentration of 300 μmol / L was added, and 30 g / L of sugar was added; 3 g / L of peracetyl bromide was added. Galactose, 15μl / LBu 4 NBr; 10 days after the medium was inoculated, the combined inducer with a concentration of 500 μmol / L was added;

[0017] (5) Extrac...

Embodiment 2

[0019] (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;

[0020] (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;

[0021] (3) Combined elicitor: composed of 0.02mol endophyte elicitor, 60g / L salicylic acid, 20μl / L hydrogen peroxide and 80μmol / L methyljasmonic acid;

[0022] (4) Combined induction and saccharification reaction: During the cultivation process, the inducer was used to cultivate, and 7 days after inoculation of the medium, the inducer with a concentration of 600 μmol / L was added, and 30 g / L of sugar was added; 6 g / L of peracetyl bromide was added. Galactose, 15μl / LBu 4 10 days after NBr was inoculated into the culture medium, a combined inducer with a concentration of 1000...

Embodiment 3

[0025] (1) Culture medium preparation: add naphthaleneacetic acid 80μmol / L, 6-benzylaminopurine 5μmol / L, sucrose or glucose 30g / L, agar 1.5g / L to MS basic medium; pH5.5~6;

[0026] (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;

[0027] (3) Combined elicitor: composed of 0.03mol endophyte elicitor, 70g / L salicylic acid, 20μl / L hydrogen peroxide and 80μmol / L methyljasmonic acid;

[0028] (4) Combined induction and saccharification reaction: During the cultivation process, the inducer was used to cultivate, and 5 days after inoculation of the medium, the inducer with a concentration of 300 μmol / L was added, and 30 g / L of sugar was added; Galactose, 15μl / LBu 4 NBr; 10 days after the medium was inoculated, the combined inducer with a concentration of 500 μmol / L was added;

[0029] (5) Extraction: The obtained culture is separated, and the cam...

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Abstract

The invention relates to a technical process for synthesizing a camptothecin sugar derivative by artificially inducing nothapodytes nimmoniana (graham) mablerley endophyte. The process disclosed by the invention comprises the following steps of: charging immature cells of nothapodytes nimmoniana (graham) mablerley into an amplification culture medium according to the proportion of inoculation amount of 50-200g / L wet weight and culturing at 25-30 DEG C; adding 1-2,000 mummol / L of combined elicitor and 20-50g / L of sugar in the exponential growth initial period of the cells; adding a saccharified reagent and catalyst for carrying out saccharification reaction; and adding 1-2,000 mummol / L of combined elicitor for inducing in the exponential growth metaphase of the cells. The method disclosed by the invention realizes the synthesis of the camptothecin derivative in an artificial inducing way by utilizing the characteristics of the endophyte of the nothapodytes nimmoniana (graham) mablerley which is the camptothecin producing plant and cansolve the problems of lower conversion rate, high technical requirement on strain culture, more separating steps, and the like existing in the traditional biosynthesis methods and provide reference for the research of camptothecin as well as the anticancer effect and anticancer medicines thereof.

Description

technical field [0001] The invention relates to a technical method for synthesizing camptothecin derivatives, in particular to a technical method for artificially inducing endophytes to synthesize camptothecin sugar derivatives, belonging to the field of biotechnology. Background technique [0002] Camptothecin (CPT) is a natural alkaloid isolated from Camptotheca involucrata. Camptothecin and its derivatives have a unique mechanism of action—the only one that can selectively inhibit DNA topoisomerase I (Topo I), so it has become one of the hot spots in antitumor drug research in the past 20 years. Due to the poor water solubility and fat solubility of camptothecin, the anticancer activity of its sodium salt is low, and its clinical application has been strictly limited. Later, people isolated 10-hydroxycamptothecin from camptothecin, and synthesized camptothecin derivatives with less toxicity and stronger anti-tumor effect, which played an important role in the prevention ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/18
Inventor 张苑金
Owner 张苑金
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