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Extraction method of Homoharringtonine through microbial fermentation and application of Homoharringtonine

A technology for homoharringtonine and microbial fermentation, which is applied in the field of bioengineering to achieve the effect of facilitating purification and treatment

Active Publication Date: 2015-07-01
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, how to efficiently extract high-purity harringtonine from the cells and fermentation broth remains to be solved

Method used

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  • Extraction method of Homoharringtonine through microbial fermentation and application of Homoharringtonine
  • Extraction method of Homoharringtonine through microbial fermentation and application of Homoharringtonine
  • Extraction method of Homoharringtonine through microbial fermentation and application of Homoharringtonine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Embodiment 1 microbial fermentation

[0083] Taking Alternaria tentoides CH1307 (preservation number CGMCC No.4443) as an example, Example 1 of the present invention provides a method for producing homoharringtonine by microbial fermentation, specifically referring to the inventor's authorized patent (Application No. : 201110028195.7) "A method for preparing homoharringtonine and a special strain" on page 8 of Example 2 (method for preparing homoharringtonine) 1-3 steps; obtain Alternaria tenopolaris CH1307 fermentation product.

Embodiment 2

[0084] Example 2 Purification of Homoharringtonine from the Fermentation Broth Removed from Bacteria

[0085] (1) Centrifuge and suction-filter the fermented material of Alternaria tenopolaris CH1307 in Example 1 to obtain 10 L of fermented liquid from which mycelium has been removed, concentrate it to 400 mL by rotary evaporation at 45° C., and adjust the pH to 8.0. Extract with chloroform four times, 100 mL each time, combine the organic phases and spin dry, redissolve in 0.05 mol / L, pH 9.0 ammonia-ammonium chloride buffer solution 100 mL.

[0086](2) The reconstituted extract was stirred and adsorbed with 10 g of NKA-9 macroporous resin for 1 h, filtered and washed twice with a small amount of the above-mentioned ammonia-ammonium chloride buffer solution. The resin was placed in 0.1mol / L pH 5.0 citric acid buffer solution:methanol (3:7) for desorption for 2h, filtered and rinsed twice with a small amount of the above desorption solution, the combined filtrate was concentrat...

Embodiment 3

[0089] Example 3 Purification of Homoharringtonine from Fermentation Broth Removed from Bacteria

[0090] (1) Centrifuge and suction-filter the fermented product of Alternaria tenopolaris CH1307 in Example 1 to obtain 20 L of fermentation broth from which mycelium has been removed, concentrate it to 500 mL by rotary evaporation at 45° C., and adjust the pH to 8.0. Extract 4 times with chloroform, 125 mL each time, combine the organic phases and spin dry, and redissolve in 200 mL of 0.05 mol / L, pH 9.0 ammonia-ammonium chloride buffer solution.

[0091] (2) The reconstituted extract was statically adsorbed with 20 g of NKA-9 macroporous resin for 1 hour, filtered and washed twice with a small amount of the above-mentioned ammonia-ammonium chloride buffer solution. The resin was placed in 0.1mol / L pH 5.0 citric acid buffer solution:methanol (3:7) for desorption for 2h, filtered and washed twice with a small amount of the above desorption solution, the combined filtrate was concen...

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Abstract

The invention provides an extraction method of Homoharringtonine through microbial fermentation and application of Homoharringtonine. The extraction method of Homoharringtonine comprises the following steps: (1) separating a microbial fermentation product to obtain thalluses and fermentation broth of which thallus is removed and carrying out organic solvent extraction and redissolution on the fermentation broth of which thallus is removed or thalluses to obtain crude extract liquid; (2) adsorbing the crude extract liquid with macroporous resin or activated carbon and carrying out desorption to obtain an alkaloid crude extract; and (3) performing high performance liquid purification on the obtained alkaloid crude extract to obtain Homoharringtonine; or firstly performing column chromatography on silica gel for purifying the obtained alkaloid crude extract to obtain crude Homoharringtonine and then performing high performance liquid phase purification on the obtained crude Homoharringtonine to obtain Homoharringtonine. According to the extraction method, since a microbial fermentation product is used as raw material, the cost is low; the process is simple and microbial thalluses or fermentation broth can be purified to obtain Homoharringtonine with high efficiency and high yield.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to microbial fermentation, a method for extracting, separating and purifying fermentation products, in particular to a method for extracting homoharringtonine from microbial fermentation and its application. Background technique [0002] Homoharringtonine (HHT) is a natural alkaloid isolated from Cephalotaxus plants with antitumor activity. Due to its extensive anti-tumor activity, homoharringtonine and its derivatives have been used in clinical research and treatment of various tumors such as leukemia, breast cancer, and liver cancer, and have shown good curative effects. In 2012, the artificial semi-synthetic homoharringtonine-Omacetaxine Mepesuccinate was approved by the US FDA for the treatment of human chronic myelogenous leukemia. [0003] At present, homoharringtonine commercially used is mainly synthetic / semi-synthetic or extracted from the harringtonine plant. Due to the slo...

Claims

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Application Information

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IPC IPC(8): C07D491/20A61P35/00
CPCC07D491/20
Inventor 李武李从发刘四新胡晓苹
Owner HAINAN UNIVERSITY
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